Methods to diagnose and treat acute respiratory infections

ABSTRACT

The present disclosure provides methods for determining the etiology of an acute respiratory infection in a subject and methods of treating the subject based on the determination, as well as systems useful for performing the determination using a biological sample from the subject.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/187,683, filed Jul. 1, 2015, and U.S. Provisional Patent Application Ser. No. 62/257,406, filed Nov. 19, 2015, the disclosure of each of which is incorporated by reference herein in its entirety.

FEDERAL FUNDING LEGEND

This invention was made with Government Support under Federal Grant Nos. U01AI066569, P20RR016480 and HHSN266200400064C awarded by the National Institutes of Health (NIH) and Federal Grant Nos. N66001-07-C-2024 and N66001-09-C-2082 awarded by the Defense Advanced Research Projects Agency (DARPA). The U.S. Government has certain rights to this invention.

BACKGROUND

Acute respiratory infection is common in acute care environments and results in significant mortality, morbidity, and economic losses worldwide. Respiratory tract infections, or acute respiratory infections (ARI) caused 3.2 million deaths around the world and 164 million disability-adjusted life years lost in 2011, more than any other cause (World Health Organization., 2013a, 2013b). In 2012, the fourth leading cause of death worldwide was lower respiratory tract infections, and in low and middle income countries, where less supportive care is available, lower respiratory tract infections are the leading cause of death (WHO factsheet, accessed August 22, 2014). These illnesses are also problematic in developed countries. In the United States in 2010, the Centers for Disease Control (CDC) determined that pneumonia and influenza alone caused 15.1 deaths for every 100,000 people in the US population. The aged and children under the age of 5 years are particularly vulnerable to poor outcomes due to ARIs. For example, in 2010, pneumonia accounted for 18.3% of all deaths, or almost 1.4 million deaths, worldwide in children aged 5 years or younger.

Pneumonia and other lower respiratory tract infections can be due to many different pathogens that are primarily viral, bacterial, or less frequently fungal. Among viral pathogens, influenza is among the most notorious based on numbers of affected individuals, variable severity from season to season, and the ever-present worry about new strains causing much higher morbidity and mortality (e.g., Avian flu). However, among viral pathogens, influenza is only one of many that cause significant human disease. Respiratory Syncytial Virus (RSV) is the leading cause of hospitalization of children in developed countries during the winter months. Worldwide, about 33 million new cases of RSV infections were reported in 2005 in children under 5, with 3.4 million severe enough for hospitalization. It is estimated that this viral infection alone kills between 66,000 and 199,000 children each year. And, in the United States alone, about 10,000 deaths annually are associated with RSV infections in the over-65 population. In addition to known viral pathogens, history has shown that new and emerging infections can manifest at any time, spreading globally within days or weeks. Recent examples include SARS-coronavirus, which had a 10% mortality rate when it appeared in 2003-2004. More recently, Middle East respiratory syndrome (MERS) coronavirus continues to simmer in the Middle East and has been associated with a 30% mortality rate. Both of these infections present with respiratory symptoms and may at first be indistinguishable from any other ARI.

Although viral infections cause the majority of ARI, bacterial etiologies are also prominent especially in the context of lower respiratory tract infections. Specific causes of bacterial ARI vary geographically and by clinical context but include Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Chlamydia pneumoniae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. The identification of these pathogens relies on their growth in culture, which typically requires days and has limited sensitivity for detection of the infectious agent. Obtaining an adequate sample to test is problematic: In a study of 1669 patients with community-acquired pneumonia, only 14% of patients could provide a “good-quality” sputum sample that resulted in a positive culture (Garcia-Vazquez et al., 2004). Clinicians are aware of the limitations of these tests, which drives uncertainty and, consequently, antibacterial therapies are frequently prescribed without any confirmation of a bacterial infection.

The ability to rapidly diagnose the etiology of ARIs is an urgent global problem with far-reaching consequences at multiple levels: optimizing treatment for individual patients; epidemiological surveillance to identify and track outbreaks; and guiding appropriate use of antimicrobials to stem the rising tide of antimicrobial resistance. It has been well established that early and appropriate antimicrobial therapy improves outcomes in patients with severe infection. This in part drives the over-utilization of antimicrobial therapies. Up to 73% of ambulatory care patients with acute respiratory illness are prescribed an antibiotic, accounting for approximately 40% of all antibiotics prescribed to adults in this setting. It has, however, been estimated that only a small fraction of these patients require anti-bacterial treatment (Cantrell et al. 2003, Clin. Ther. January; 24(1):170-82). A similar trend is observed in emergency departments. Even if the presence of a viral pathogen has been microbiologically confirmed, it does not preclude the possibility of a concurrent bacterial infection. As a result, antibacterials are often prescribed “just in case.” This spiraling empiricism contributes to the rising tide of antimicrobial resistance (Gould, 2009; Kim & Gallis, 1989), which is itself associated with higher mortality, length of hospitalization, and costs of health care (Cosgrove 2006, Clin. Infect. Dis., January 15; 42 Suppl 2:S82-9). In addition, the inappropriate use of antibiotics may lead to drug-related adverse effects and other complications, e.g., Clostridium difficile-associated diarrhea (Zaas et al., 2014).

Acute respiratory infections are frequently characterized by non-specific symptoms (such as fever or cough) that are common to many different illnesses, including illnesses that are not caused by an infection. Existing diagnostics for ARI fall short in a number of ways. Conventional microbiological testing is limited by poor sensitivity and specificity, slow turn-around times, or by the complexity of the test (Zaas et al. 2014, Trends Mol Med 20(10):579-88). One limitation of current tests that detect specific viral pathogens, for example the multiplex PCR-based assays, is the inability to detect emergent or pandemic viral strains. Influenza pandemics arise when new viruses circulate against which populations have no natural resistance. Influenza pandemics are frequently devastating. For example, in 1918-1919 the Spanish flu affected about 20% to 40% of the world's population and killed about 50 million people; in 1957-1958, Asian flu killed about 2 million people; in 1968-1969 the Hong Kong flu killed about 1 million people; and in 2009-2010, the Centers for Disease Control estimates that approximately 43 million to 89 million people contracted swine flu resulting in 8,870 to 18,300 related deaths. The emergence of these new strains challenges existing diagnostics which are not designed to detect them. This was particularly evident during the 2009 influenza pandemic where confirmation of infection required days and only occurred at specialized testing centers such as state health departments or the CDC (Kumar & Henrickson 2012, Clin Microbiol Rev 25(2):344-61). The Ebola virus disease outbreak in West Africa poses similar challenges at the present time. Moreover, there is every expectation we will continue to face this issue as future outbreaks of infectious diseases are inevitable.

A further limitation of diagnostics that use the paradigm of testing for specific viruses or bacteria is that even though a pathogenic microbe may be detected, this is not proof that the patient's symptoms are due to the detected pathogen. A microorganism may be present as part of the individual's normal flora, known as colonization, or it may be detected due to contamination of the tested sample (e.g., a nasal swab or wash). Although recently-approved multiplex PCR assays, including those that detect viruses and bacteria, offer high sensitivity, these tests do not differentiate between asymptomatic carriage of a virus and true infection. For example, there is a high rate of asymptomatic viral shedding in ARI, particularly in children (Jansen et al. 2011, J Clin Microbiol 49(7):2631-2636). Similarly, even though one pathogen is detected, illness may be due to a second pathogen for which there was no test available or performed.

Reports have described host gene expression profiles differentiating viral ARI from healthy controls (Huang et al. 2011 PLoS Genetics 7(8): e1002234; Mejias et al., 2013; Thach et al. 2005 Genes and Immunity 6:588-595; Woods et al., 2013; A. K. Zaas et al., 2013; A. K. Zaas et al., 2009). However, few among these differentiate viral from bacterial ARI, which is a more clinically meaningful distinction than is detection of viral infection versus healthy or bacterial infection versus healthy (Hu, Yu, Crosby, & Storch, 2013; Parnell et al., 2012; Ramilo et al., 2007).

Current diagnostics methods are thus limited in their ability to differentiate between a bacterial and viral infection, and symptoms arising from non-infectious causes, or to identify co-infections with bacteria and virus.

SUMMARY

The present disclosure provides, in part, a molecular diagnostic test that overcomes many of the limitations of current methods for the determination of the etiology of respiratory symptoms. The test detects the host's response to an infectious agent or agents by measuring and analyzing the patterns of co-expressed genes, or signatures. These gene expression signatures may be measured in a blood sample in a human or animal presenting with symptoms that are consistent with an acute respiratory infection or in a human or animal that is at risk of developing (e.g., presymptomatic) an acute respiratory infection (e.g., during an epidemic or local disease outbreak). Measurement of the host response as taught herein differentiates between bacterial ARI, viral ARI, and a non-infectious cause of illness, and may also detect ARI resulting from co-infection with bacteria and virus.

This multi-component test performs with unprecedented accuracy and clinical applicability, allowing health care providers to use the response of the host (the subject or patient) to reliably determine the nature of the infectious agent, to the level of pathogen class, or to exclude an infectious cause of symptoms in an individual patient presenting with symptoms that, by themselves, are not specific. In some embodiments, the results are agnostic to the species of respiratory virus or bacteria (i.e., while differentiating between virus or bacteria, it does not differentiate between particular genus or species of virus or bacteria). This offers an advantage over current tests that include probes or reagents directed to specific pathogens and thus are limited to detecting only those specific pathogens.

One aspect of the present disclosure provides a method for determining whether acute respiratory symptoms in a subject are bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of: (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes, termed signatures; (c) normalizing gene expression levels for the technology (i.e., platform) used to make said measurement to generate a normalized value; (d) entering the normalized values into a bacterial classifier, a viral classifier and/or a non-infectious illness classifier that have pre-defined weighting values (coefficients) for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; and (f) using the output to determine whether the patient providing the sample has an infection of bacterial origin, viral origin, or has a non-infectious illness, or some combination of these conditions.

Another aspect of the present disclosure provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of: (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes; (c) normalizing gene expression levels for the technology (i.e., platform) used to make said measurement to generate a normalized value; (d) entering the normalized value into classifiers that have pre-defined weighting values for each of the genes in each signature; e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) if the sample is negative for bacteria, repeating step (d) using only the viral classifier and non-infectious classifier; and (g) classifying the sample as being of viral etiology or noninfectious illness.

Another aspect of the present disclosure provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of: (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes; (c) normalizing gene expression levels for the technology (i.e., platform) used to make said measurement to generate a normalized value; (d) entering the normalized values into classifiers that have pre-defined weighting values for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) if the sample is negative for virus, repeating step (d) using only the bacteria classifier and non-infectious classifier; and (g) classifying the sample as being of bacterial etiology or noninfectious illness.

Another aspect of the present disclosure provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of: (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes; (c) normalizing gene expression levels for the technology (i.e., platform) used to make said measurement to generate a normalized value; (d) entering the normalized values into classifiers that have pre-defined weighting values for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) if the sample is negative for non-infectious illness, repeating step (d) using only the viral classifier and bacterial classifier; and (g) classifying the sample as being of viral etiology or bacterial etiology.

Yet another aspect of the present disclosure provides a method of treating an acute respiratory infection (ARI) whose etiology is unknown in a subject, said method comprising, consisting of, or consisting essentially of: (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes (e.g., one, two or three or more signatures); (c) normalizing gene expression levels for the technology (i.e., platform) used to make said measurement to generate a normalized value; (d) entering the normalized values into a bacterial classifier, a viral classifier and non-infectious illness classifier that have pre-defined weighting values for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) classifying the sample as being of bacterial etiology, viral etiology, or noninfectious illness; and (g) administering to the subject an appropriate treatment regimen as identified by step (e). In some embodiments, step (g) comprises administering an antibacterial therapy when the etiology of the ARI is determined to be bacterial. In other embodiments, step (g) comprises administering an antiviral therapy when the etiology of the ARI is determined to be viral.

Another aspect is a method of monitoring response to a vaccine or a drug in a subject suffering from or at risk of an acute respiratory illness selected from bacterial, viral and/or non-infectious, comprising determining a host response of said subject, said determining carried out by a method as taught herein. In some embodiments, the drug is an antibacterial drug or an antiviral drug.

In some embodiments of the aspects, the methods further comprise generating a report assigning the subject a score indicating the probability of the etiology of the ARI.

Further provided is a system for determining an etiology of an acute respiratory illness in a subject selected from bacterial, viral and/or non-infectious, comprising one or more of (inclusive of combinations thereof): at least one processor; a sample input circuit configured to receive a biological sample from the subject; a sample analysis circuit coupled to the at least one processor and configured to determine gene expression levels of the biological sample; an input/output circuit coupled to the at least one processor; a storage circuit coupled to the at least one processor and configured to store data, parameters, and/or classifiers; and a memory coupled to the processor and comprising computer readable program code embodied in the memory that when executed by the at least one processor causes the at least one processor to perform operations comprising: controlling/performing measurement via the sample analysis circuit of gene expression levels of a pre-defined set of genes (i.e., signature) in said biological sample; normalizing the gene expression levels to generate normalized gene expression values; retrieving from the storage circuit a bacterial acute respiratory infection (ARI) classifier, a viral ARI classifier and a non-infectious illness classifier, said classifier(s) comprising pre-defined weighting values (i.e., coefficients) for each of the genes of the pre-defined set of genes; entering the normalized gene expression values into one or more acute respiratory illness classifiers selected from the bacterial acute respiratory infection (ARI) classifier, the viral ARI classifier and the non-infectious illness classifier; calculating an etiology probability for one or more of a bacterial ARI, viral ARI and non-infectious illness based upon said classifier(s); and controlling output via the input/output circuit of a determination whether the acute respiratory illness in the subject is bacterial in origin, viral in origin, non-infectious in origin, or some combination thereof.

In some embodiments, the system comprises computer readable code to transform quantitative, or semi-quantitative, detection of gene expression to a cumulative score or probability of the etiology of the ARI.

In some embodiments, the system comprises an array platform, a thermal cycler platform (e.g., multiplexed and/or real-time PCR platform), a hybridization and multi-signal coded (e.g., fluorescence) detector platform, a nucleic acid mass spectrometry platform, a nucleic acid sequencing platform, or a combination thereof.

In some embodiments of the aspects, the pre-defined sets of genes comprise at least three genetic signatures.

In some embodiments of the aspects, the biological sample comprises a sample selected from the group consisting of peripheral blood, sputum, nasopharyngeal swab, nasopharyngeal wash, bronchoalveolar lavage, endotracheal aspirate, and combinations thereof.

In some embodiments of the aspects, the bacterial classifier comprises expression levels of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes (measurable, e.g., with oligonucleotide probes homologous to said genes or gene transcripts) listed as part of a bacterial classifier in Table 1, Table 2, Table 9, Table 10 and/or Table 12. In some embodiments, the viral classifier comprises expression levels of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes (measurable, e.g., with oligonucleotide probes homologous to said genes or gene transcripts) listed as part of a viral classifier in Table 1, Table 2, Table 9, Table 10 and/or Table 12. In some embodiments, the non-infectious illness classifier comprises expression levels of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes (measurable, e.g., with oligonucleotide probes homologous to said genes or gene transcripts) listed as part of a non-infectious illness classifier in Table 1, Table 2, Table 9, Table 10 and/or Table 12.

A kit for determining the etiology of an acute respiratory infection (ARI) in a subject is also provided, comprising, consisting of, or consisting essentially of (a) a means for extracting mRNA from a biological sample; (b) a means for generating one or more arrays consisting of a plurality of synthetic oligonucleotides with regions homologous to transcripts from of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes from Table 1, Table 2, Table 9, Table 10 and/or Table 12; and (c) instructions for use.

Another aspect of the present disclosure provides a method of using a kit for assessing the acute respiratory infection (ARI) classifier comprising, consisting of, or consisting essentially of: (a) generating one or more arrays consisting of a plurality of synthetic oligonucleotides with regions homologous to of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes from Table 1, Table 2, Table 9, Table 10 and/or Table 12; (b) adding to said array oligonucleotides with regions homologous to normalizing genes; (c) obtaining a biological sample from a subject suffering from an acute respiratory infection (ARI); (d) isolating RNA from said sample to create a transcriptome; (e) measuring said transcriptome on said array (e.g., by measuring fluorescence or electric current proportional to the level of gene expression, etc.); (f) normalizing the measurements of said transcriptome to the normalizing genes, electronically transferring normalized measurements to a computer to implement the classifier(s), (g) generating a report; and optionally (h) administering an appropriate treatment based on the results.

In some embodiments, the method further comprises externally validating an ARI classifier against a known dataset comprising at least two relevant clinical attributes. In some embodiments, the dataset is selected from the group consisting of GSE6269, GSE42026, GSE40396, GSE20346, GSE42834 and combinations thereof.

Yet another aspect of the present disclosure provides all that is disclosed and illustrated herein.

Also provided is the use of an ARI classifier as taught herein in a method of treatment for acute respiratory infection (ARI) in a subject of unknown etiology.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying drawings, herein:

FIG. 1 is a schematic showing a method of obtaining classifiers (training 10) according to some embodiments of the present disclosure, where each classifier is composed of a weighted sum of all or a subset of normalized gene expression levels. This weighted sum defines a probability that allows for a decision (classification), particularly when compared to a threshold value or a confidence interval. The exact combination of genes, their weights and the threshold for each classifier obtained by the training are particular to a specific platform. The classifier (or more precisely its components, namely weights and threshold or confidence interval (values)) go to a database. Weights with a nonzero value determine the subset of genes used by the classifier. Repeat to obtain all three classifiers (bacterial ARI, viral ARI and non-infectious ARI) within a specified platform matching the gene expression values.

FIG. 2 is a diagram showing an example of generating and/or using classifiers in accordance with some embodiments of the present disclosure.

FIG. 3 is a schematic showing a method of classification 20 of an etiology of acute respiratory symptoms suffered by a subject making use of classifiers according to some embodiments of the present disclosure.

FIG. 4 presents schematics showing the decision pattern for using secondary classification to determine the etiology of an ARI in a subject in accordance with some embodiments of the present disclosure.

FIG. 5 is a diagram of an example training method presented in Example 1. A cohort of patients encompassing bacterial ARI, viral ARI, or non-infectious illness was used to develop classifiers of each condition. This combined ARI classifier was validated using leave one out cross-validation and compared to three published classifiers of bacterial vs. viral infection. The combined ARI classifier was also externally validated in six publically available datasets. In one experiment, healthy volunteers were included in the training set to determine their suitability as “no-infection” controls. All subsequent experiments were performed without the use of this healthy subject cohort.

FIG. 6 presents graphs showing the results of leave-one-out cross-validation of three classifiers (bacterial ARI, viral ARI and noninfectious illness) according an example training method presented in Example 1. Each patient is assigned probabilities of having bacterial ARI (triangle), viral ARI (circle), and non-infectious illness (square). Patients clinically adjudicated as having bacterial ARI, viral ARI, or non-infectious illness, are presented in the top, center, and bottom panels, respectively. Overall classification accuracy was 87%.

FIG. 7 is a graph showing the evaluation of healthy adults as a no-infection control, rather than an ill-but-uninfected control. This figure demonstrates the unexpected superiority of the use of ill-but-not infected subjects as the control.

FIG. 8 shows the positive and negative predictive values for A) Bacterial and B) Viral ARI classification as a function of prevalence.

FIG. 9 is a Venn diagram representing overlap in the Bacterial ARI, Viral ARI, and Non-infectious Illness Classifiers. There are 71 genes in the Bacterial ARI Classifier, 33 in the Viral

ARI Classifier, and 26 in the Non-infectious Illness Classifier. One gene overlaps between the Bacterial and Viral ARI Classifiers. Five genes overlap between the Bacterial ARI and Non-infectious Illness Classifiers. Four genes overlap between the Viral ARI and Non-infectious Illness Classifiers.

FIG. 10 is a graph showing Classifier performance in patients with co-infection by the identification of bacterial and viral pathogens. Bacterial and Viral ARI classifiers were trained on subjects with bacterial (n=22) or viral (n=71) infection (GSE60244). This same dataset also included 25 subjects with bacterial/viral co-infection. Bacterial and viral classifier predictions were normalized to the same scale, as shown in the figure. Each subject receives two probabilities: that of a bacterial ARI host response and a viral ARI host response. A probability score of 0.5 or greater was considered positive. Subjects 1-6 have a bacterial host response. Subjects 7-9 have both bacterial and viral host responses which may indicate true co-infection. Subjects 10-23 have a viral host response. Subjects 24-25 do not have bacterial or viral host responses.

FIG. 11 is a block diagram of a classification system and/or computer program product that may be used in a platform. A classification system and/or computer program product 1100 may include a processor subsystem 1140, including one or more Central Processing Units (CPU) on which one or more operating systems and/or one or more applications run. While one processor 1140 is shown, it will be understood that multiple processors 1140 may be present, which may be either electrically interconnected or separate. Processor(s) 1140 are configured to execute computer program code from memory devices, such as memory 1150, to perform at least some of the operations and methods described herein. The storage circuit 1170 may store databases which provide access to the data/parameters/classifiers used by the classification system 1110 such as the signatures, weights, thresholds, etc. An input/output circuit 1160 may include displays and/or user input devices, such as keyboards, touch screens and/or pointing devices. Devices attached to the input/output circuit 1160 may be used to provide information to the processor 1140 by a user of the classification system 1100. Devices attached to the input/output circuit 1160 may include networking or communication controllers, input devices (keyboard, a mouse, touch screen, etc.) and output devices (printer or display). An optional update circuit 1180 may be included as an interface for providing updates to the classification system 1100 such as updates to the code executed by the processor 1140 that are stored in the memory 1150 and/or the storage circuit 1170. Updates provided via the update circuit 1180 may also include updates to portions of the storage circuit 1170 related to a database and/or other data storage format which maintains information for the classification system 1100, such as the signatures, weights, thresholds, etc. The sample input circuit 1110 provides an interface for the classification system 1100 to receive biological samples to be analyzed. The sample processing circuit 1120 may further process the biological sample within the classification system 1100 so as to prepare the biological sample for automated analysis.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The present disclosure provides that alterations in gene, protein and metabolite expression in blood in response to pathogen exposure that causes acute respiratory infections can be used to identify and characterize the etiology of the ARI in a subject with a high degree of accuracy.

Definitions

As used herein, the term “acute respiratory infection” or “ARI” refers to an infection, or an illness showing symptoms and/or physical findings consistent with an infection (e.g., symptoms such as coughing, wheezing, fever, sore throat, congestion; physical findings such as elevated heart rate, elevated breath rate, abnormal white blood cell count, low arterial carbon dioxide tension (PaCO₂), etc.), of the upper or lower respiratory tract, often due to a bacterial or viral pathogen, and characterized by rapid progression of symptoms over hours to days. ARIs may primarily be of the upper respiratory tract (URIs), the lower respiratory tract (LRIs), or a combination of the two. ARIs may have systemic effects due to spread of the infection beyond the respiratory tract or due to collateral damage induced by the immune response. An example of the former includes Staphylococcus aureus pneumonia that has spread to the blood stream and can result in secondary sites of infection, including endocarditis (infection of the heart valves), septic arthritis (joint infection), or osteomyelitis (bone infection). An example of the latter includes influenza pneumonia leading to acute respiratory distress syndrome and respiratory failure.

The term “signature” as used herein refers to a set of biological analytes and the measurable quantities of said analytes whose particular combination signifies the presence or absence of the specified biological state. These signatures are discovered in a plurality of subjects with known status (e.g., with a confirmed respiratory bacterial infection, respiratory viral infection, or suffering from non-infectious illness), and are discriminative (individually or jointly) of one or more categories or outcomes of interest. These measurable analytes, also known as biological markers, can be (but are not limited to) gene expression levels, protein or peptide levels, or metabolite levels. See also US 2015/0227681 to Courchesne et al.; US 2016/0153993 to Eden et al.

In some embodiments as disclosed herein, the “signature” is a particular combination of genes whose expression levels, when incorporated into a classifier as taught herein, discriminate a condition such as a bacterial ARI, viral ARI or non-infectious illness. See, for example, Table 1, Table 2, Table 9, Table 10 and Table 12 hereinbelow. In some embodiments, the signature is agnostic to the species of respiratory virus or bacteria (i.e., while differentiating between virus or bacteria, it does not differentiate between particular genus or species of virus or bacteria) and/or agnostic to the particular cause of the non-infectious illness.

As used herein, the terms “classifier” and “predictor” are used interchangeably and refer to a mathematical function that uses the values of the signature (e.g., gene expression levels for a defined set of genes) and a pre-determined coefficient (or weight) for each signature component to generate scores for a given observation or individual patient for the purpose of assignment to a category. The classifier may be linear and/or probabilistic. A classifier is linear if scores are a function of summed signature values weighted by a set of coefficients. Furthermore, a classifier is probabilistic if the function of signature values generates a probability, a value between 0 and 1.0 (or 0 and 100%) quantifying the likelihood that a subject or observation belongs to a particular category or will have a particular outcome, respectively. Probit regression and logistic regression are examples of probabilistic linear classifiers that use probit and logistic link functions, respectively, to generate a probability.

A classifier as taught herein may be obtained by a procedure known as “training,” which makes use of a set of data containing observations with known category membership (e.g., bacterial ARI, viral ARI, and/or non-infection illness). See FIG. 1. Specifically, training seeks to find the optimal coefficient (i.e., weight) for each component of a given signature (e.g., gene expression level components), as well as an optimal signature, where the optimal result is determined by the highest achievable classification accuracy.

“Classification” refers to a method of assigning a subject suffering from or at risk for acute respiratory symptoms to one or more categories or outcomes (e.g., a patient is infected with a pathogen or is not infected, another categorization may be that a patient is infected with a virus and/or infected with a bacterium). See FIG. 3. In some cases, a subject may be classified to more than one category, e.g., in case of bacterial and viral co-infection. The outcome, or category, is determined by the value of the scores provided by the classifier, which may be compared to a cut-off or threshold value, confidence level, or limit. In other scenarios, the probability of belonging to a particular category may be given (e.g., if the classifier reports probabilities).

As used herein, the term “indicative” when used with gene expression levels, means that the gene expression levels are up-regulated or down-regulated, altered, or changed compared to the expression levels in alternative biological states (e.g., bacterial ARI or viral ARI) or control. The term “indicative” when used with protein levels means that the protein levels are higher or lower, increased or decreased, altered, or changed compared to the standard protein levels or levels in alternative biological states.

The term “subject” and “patient” are used interchangeably and refer to any animal being examined, studied or treated. It is not intended that the present disclosure be limited to any particular type of subject. In some embodiments of the present invention, humans are the preferred subject, while in other embodiments non-human animals are the preferred subject, including, but not limited to, mice, monkeys, ferrets, cattle, sheep, goats, pigs, chicken, turkeys, dogs, cats, horses and reptiles. In certain embodiments, the subject is suffering from an ARI or is displaying ARI-like symptoms.

“Platform” or “technology” as used herein refers to an apparatus (e.g., instrument and associated parts, computer, computer-readable media comprising one or more databases as taught herein, reagents, etc.) that may be used to measure a signature, e.g., gene expression levels, in accordance with the present disclosure. Examples of platforms include, but are not limited to, an array platform, a thermal cycler platform (e.g., multiplexed and/or real-time PCR platform), a nucleic acid sequencing platform, a hybridization and multi-signal coded (e.g., fluorescence) detector platform, etc., a nucleic acid mass spectrometry platform, a magnetic resonance platform, and combinations thereof.

In some embodiments, the platform is configured to measure gene expression levels semi-quantitatively, that is, rather than measuring in discrete or absolute expression, the expression levels are measured as an estimate and/or relative to each other or a specified marker or markers (e.g., expression of another, “standard” or “reference,” gene).

In some embodiments, semi-quantitative measuring includes “real-time PCR” by performing PCR cycles until a signal indicating the specified mRNA is detected, and using the number of PCR cycles needed until detection to provide the estimated or relative expression levels of the genes within the signature.

A real-time PCR platform includes, for example, a TaqMan® Low Density Array (TLDA), in which samples undergo multiplexed reverse transcription, followed by real-time PCR on an array card with a collection of wells in which real-time PCR is performed. See Kodani et al. 2011, J. Clin. Microbial. 49(6):2175-2182. A real-time PCR platform also includes, for example, a Biocartis Idylla™ sample-to-result technology, in which cells are lysed, DNA/RNA extracted and real-time PCR is performed and results detected.

A magnetic resonance platform includes, for example, T2 Biosystems® T2 Magnetic Resonance (T2MR®) technology, in which molecular targets may be identified in biological samples without the need for purification.

The terms “array,” “microarray” and “micro array” are interchangeable and refer to an arrangement of a collection of nucleotide sequences presented on a substrate. Any type of array can be utilized in the methods provided herein. For example, arrays can be on a solid substrate (a solid phase array), such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane. Arrays can also be presented on beads, i.e., a bead array. These beads are typically microscopic and may be made of, e.g., polystyrene. The array can also be presented on nanoparticles, which may be made of, e.g., particularly gold, but also silver, palladium, or platinum. See, e.g., Nanosphere Verigene® System, which uses gold nanoparticle probe technology. Magnetic nanoparticles may also be used. Other examples include nuclear magnetic resonance microcoils. The nucleotide sequences can be DNA, RNA, or any permutations thereof (e.g., nucleotide analogues, such as locked nucleic acids (LNAs), and the like). In some embodiments, the nucleotide sequences span exon/intron boundaries to detect gene expression of spliced or mature RNA species rather than genomic DNA. The nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences. The arrays may additionally comprise other compounds, such as antibodies, peptides, proteins, tissues, cells, chemicals, carbohydrates, and the like that specifically bind proteins or metabolites.

An array platform includes, for example, the TaqMan® Low Density Array (TLDA) mentioned above, and an Affymetrix® microarray platform.

A hybridization and multi-signal coded detector platform includes, for example, NanoString nCounter® technology, in which hybridization of a color-coded barcode attached to a target-specific probe (e.g., corresponding to a gene expression transcript of interest) is detected; and Luminex® xMAP® technology, in which microsphere beads are color coded and coated with a target-specific (e.g., gene expression transcript) probe for detection; and Illumina® BeadArray, in which microbeads are assembled onto fiber optic bundles or planar silica slides and coated with a target-specific (e.g., gene expression transcript) probe for detection.

A nucleic acid mass spectrometry platform includes, for example, the Ibis Biosciences Plex-ID® Detector, in which DNA mass spectrometry is used to detect amplified DNA using mass profiles.

A thermal cycler platform includes, for example, the FilmArray® multiplex PCR system, which extract and purifies nucleic acids from an unprocessed sample and performs nested multiplex PCR; and the RainDrop Digital PCR System, which is a droplet-based PCR platform using microfluidic chips.

The term “computer readable medium” refers to any device or system for storing and providing information (e.g., data and instructions) to a computer processor. Examples of computer readable media include, but are not limited to, DVDs, CDs hard disk drives, magnetic tape and servers for streaming media over networks, and applications, such as those found on smart phones and tablets. In various embodiments, aspects of the present invention including data structures and methods may be stored on a computer readable medium. Processing and data may also be performed on numerous device types, including but not limited to, desk top and lap top computers, tablets, smart phones, and the like.

As used herein, the term “biological sample” comprises any sample that may be taken from a subject that contains genetic material that can be used in the methods provided herein. For example, a biological sample may comprise a peripheral blood sample. The term “peripheral blood sample” refers to a sample of blood circulating in the circulatory system or body taken from the system of body. Other samples may comprise those taken from the upper respiratory tract, including but not limited to, sputum, nasopharyngeal swab and nasopharyngeal wash. A biological sample may also comprise those samples taken from the lower respiratory tract, including but not limited to, bronchoalveolar lavage and endotracheal aspirate. A biological sample may also comprise any combinations thereof.

The term “genetic material” refers to a material used to store genetic information in the nuclei or mitochondria of an organism's cells. Examples of genetic material include, but are not limited to, double-stranded and single-stranded DNA, cDNA, RNA, and mRNA.

The term “plurality of nucleic acid oligomers” refers to two or more nucleic acid oligomers, which can be DNA or RNA.

As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the severity, duration and/or progression of a disease or disorder or one or more symptoms thereof resulting from the administration of one or more therapies. Such terms refer to a reduction in the replication of a virus or bacteria, or a reduction in the spread of a virus or bacteria to other organs or tissues in a subject or to other subjects. Treatment may also include therapies for ARIs resulting from non-infectious illness, such as allergy treatment, asthma treatments, and the like.

The term “effective amount” refers to an amount of a therapeutic agent that is sufficient to exert a physiological effect in the subject. The term “responsivity” refers to a change in gene expression levels of genes in a subject in response to the subject being infected with a virus or bacteria or suffering from a non-infectious illness compared to the gene expression levels of the genes in a subject that is not infected with a virus, bacteria or suffering from a non-infectious illness or a control subject.

The term “appropriate treatment regimen” refers to the standard of care needed to treat a specific disease or disorder. Often such regimens require the act of administering to a subject a therapeutic agent(s) capable of producing a curative effect in a disease state. For example, a therapeutic agent for treating a subject having bacteremia is an antibiotic which include, but are not limited to, penicillins, cephalosporins, fluroquinolones, tetracyclines, macrolides, and aminoglycosides. A therapeutic agent for treating a subject having a viral respiratory infection includes, but is not limited to, oseltamivir, RNAi antivirals, inhaled ribavirin, monoclonal antibody respigam, zanamivir, and neuraminidase blocking agents. The invention contemplates the use of the methods of the invention to determine treatments with antivirals or antibiotics that are not yet available. Appropriate treatment regimes also include treatments for ARIs resulting from non-infectious illness, such as allergy treatments, including but not limited to, administration of antihistamines, decongestants, anticholinergic nasal sprays, leukotriene inhibitors, mast cell inhibitors, steroid nasal sprays, etc.; and asthma treatments, including, but not limited to, inhaled corticosteroids, leukotriene modifiers, long-acting beta agonists, combinations inhalers (e.g., fluticasone-salmeterol; budesonide-formoterol; mometasone-formoterol, etc.), theophylline, short-acting beta agonists, ipratropium, oral and intravenous corticosteroids, omalizumab, and the like.

Often such regimens require the act of administering to a subject a therapeutic agent(s) capable of producing reduction of symptoms associated with a disease state. Examples such therapeutic agents include, but are not limited to, NSAIDS, acetaminophen, anti-histamines, beta-agonists, anti-tussives or other medicaments that reduce the symptoms associated with the disease process.

Methods of Generating Classifiers (Training)

The present disclosure provides methods of generating classifiers (also referred to as training 10) for use in the methods of determining the etiology of an acute respiratory illness in a subject. Gene expression-based classifiers are developed that can be used to identify and characterize the etiology of an ARI in a subject with a high degree of accuracy.

Hence, and as shown in FIG. 1, one aspect of the present disclosure provides a method of making an acute respiratory infection (ARI) classifier comprising, consisting of, or consisting essentially of: (i) obtaining a biological sample (e.g., a peripheral blood sample) from a plurality of subjects suffering from bacterial, viral or non-infectious acute respiratory infection 100; (ii) optionally, isolating RNA from said sample (e.g., total RNA to create a transcriptome) (105, not shown in FIG. 1); (iii) measuring gene expression levels of a plurality of genes 110 (i.e., some or all of the genes expressed in the RNA); (iv) normalizing the gene expression levels 120; and (v) generating a bacterial ARI classifier, a viral ARI classifier or a non-infectious illness classifier 130 based on the results.

In some embodiments, the sample is not purified after collection. In some embodiments, the sample may be purified to remove extraneous material, before or after lysis of cells. In some embodiments, the sample is purified with cell lysis and removal of cellular materials, isolation of nucleic acids, and/or reduction of abundant transcripts such as globin or ribosomal RNAs.

In some embodiments, measuring gene expression levels may include generating one or more microarrays using said transcriptomes; measuring said transcriptomes using a plurality of primers; analyzing and correcting batch differences.

In some embodiments, the method further includes uploading 140 the final gene target list for the generated classifier, the associated weights (w_(n)), and threshold values to one or more databases.

An example of generating said classifiers is detailed in FIG. 2. As shown in FIG. 2, biological samples from a cohort of patients encompassing bacterial ARI, viral ARI, or non-infectious illness are used to develop gene expression-based classifiers for each condition (i.e., bacterial acute respiratory infection, viral acute respiratory infection, or non-infectious cause of illness). Specifically, the bacterial ARI classifier is obtained to positively identifying those with bacterial ARI vs. either viral ARI or non-infectious illnesses. The viral ARI classifier is obtained to positively identifying those with viral ARI vs. bacterial ARI or non-infectious illness (NI). The non-infectious illness classifier is generated to improve bacterial and viral ARI classifier specificity. Next, signatures for bacterial ARI classifiers, viral ARI classifiers, and non-infectious illness classifiers are generated (e.g., by applying a sparse logistic regression model).

These three classifiers may then be combined, if desired, into a single classifier termed “the ARI classifier” by following a one-versus-all scheme whereby largest membership probability assigns class label. See also FIG. 5. The combined ARI classifier may be validated in some embodiments using leave-one-out cross-validation in the same population from which it was derived and/or may be validated in some embodiments using publically available human gene expression datasets of samples from subjects suffering from illness of known etiology. For example, validation may be performed using publically available human gene expression datasets (e.g., GSE6269, GSE42026, GSE40396, GSE20346, and/or GSE42834), the datasets chosen if they included at least two clinical groups (bacterial ARI, viral ARI, or non-infectious illness).

The classifier may be validated in a standard set of samples from subjects suffering from illness of known etiology, i.e., bacterial ARI, viral ARI, or non-infectious illness.

The methodology for training described herein may be readily translated by one of ordinary skill in the art to different gene expression detection (e.g., mRNA detection and quantification) platforms.

The methods and assays of the present disclosure may be based upon gene expression, for example, through direct measurement of RNA, measurement of derived materials (e.g., cDNA), and measurement of RNA products (e.g., encoded proteins or peptides). Any method of extracting and screening gene expression may be used and is within the scope of the present disclosure.

In some embodiments, the measuring comprises the detection and quantification (e.g., semi-quantification) of mRNA in the sample. In some embodiments, the gene expression levels are adjusted relative to one or more standard gene level(s) (“normalized”). As known in the art, normalizing is done to remove technical variability inherent to a platform to give a quantity or relative quantity (e.g., of expressed genes).

In some embodiments, detection and quantification of mRNA may first involve a reverse transcription and/or amplification step, e.g., RT-PCR such as quantitative RT-PCR. In some embodiments, detection and quantification may be based upon the unamplified mRNA molecules present in or purified from the biological sample. Direct detection and measurement of RNA molecules typically involves hybridization to complementary primers and/or labeled probes. Such methods include traditional northern blotting and surface-enhanced Raman spectroscopy (SERS), which involves shooting a laser at a sample exposed to surfaces of plasmonic-active metal structures with gene-specific probes, and measuring changes in light frequency as it scatters.

Similarly, detection of RNA derivatives, such as cDNA, typically involves hybridization to complementary primers and/or labeled probes. This may include high-density oligonucleotide probe arrays (e.g., solid state microarrays and bead arrays) or related probe-hybridization methods, and polymerase chain reaction (PCR)-based amplification and detection, including real-time, digital, and end-point PCR methods for relative and absolute quantitation of specific RNA molecules.

Additionally, sequencing-based methods can be used to detect and quantify RNA or

RNA-derived material levels. When applied to RNA, sequencing methods are referred to as RNAseq, and provide both qualitative (sequence, or presence/absence of an RNA, or its cognate cDNA, in a sample) and quantitative (copy number) information on RNA molecules from a sample. See, e.g., Wang et al. 2009 Nat. Rev. Genet. 10(1):57-63. Another sequence-based method, serial analysis of gene expression (SAGE), uses cDNA “tags” as a proxy to measure expression levels of RNA molecules.

Moreover, use of proprietary platforms for mRNA detection and quantification may also be used to complete the methods of the present disclosure. Examples of these are Pixel™ System, incorporating Molecular Indexing™, developed by CELLULAR RESEARCH, INC., NanoString® Technologies nCounter gene expression system; mRNA-Seq, Tag-Profiling, BeadArray™ technology and VeraCode from Illumina, the ICEPlex System from PrimeraDx, and the QuantiGene 2.0 Multiplex Assay from Affymetrix.

As an example, RNA from whole blood from a subject can be collected using RNA preservation reagents such as PAXgene™ RNA tubes (PreAnalytiX, Valencia, Calif.). The RNA can be extracted using a standard PAXgene™ or Versagene™ (Gentra Systems, Inc, Minneapolis, Minn.) RNA extraction protocol. The Versagene™ kit produces greater yields of higher quality RNA from the PAXgene™ RNA tubes. Following RNA extraction, one can use GLOBINCIear™ (Ambion, Austin, Tex.) for whole blood globin reduction. (This method uses a bead-oligonucleotide construct to bind globin mRNA and, in our experience, we are able to remove over 90% of the globin mRNA.) Depending on the technology, removal of abundant and non-interesting transcripts may increase the sensitivity of the assay, such as with a microarray platform.

Quality of the RNA can be assessed by several means. For example, RNA quality can be assessed using an Agilent 2100 Bioanalyzer immediately following extraction. This analysis provides an RNA Integrity Number (RIN) as a quantitative measure of RNA quality. Also, following globin reduction the samples can be compared to the globin-reduced standards. In addition, the scaling factors and background can be assessed following hybridization to microarrays.

Real-time PCR may be used to quickly identify gene expression from a whole blood sample. For example, the isolated RNA can be reverse transcribed and then amplified and detected in real time using non-specific fluorescent dyes that intercalate with the resulting ds-DNA, or sequence-specific DNA probes labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

Hence, it should be understood that there are many methods of mRNA quantification and detection that may be used by a platform in accordance with the methods disclosed herein.

The expression levels are typically normalized following detection and quantification as appropriate for the particular platform using methods routinely practiced by those of ordinary skill in the art.

With mRNA detection and quantification and a matched normalization methodology in place for platform, it is simply a matter of using carefully selected and adjudicated patient samples for the training methods. For example, the cohort described hereinbelow was used to generate the appropriate weighting values (coefficients) to be used in conjunction with the genes in the three signatures in the classifier for a platform. These subject-samples could also be used to generate coefficients and cut-offs for a test implemented using a different mRNA detection and quantification platform.

In some embodiments, the individual categories of classifiers (i.e., bacterial ARI, viral ARI, non-infectious illness) are formed from a cohort inclusive of a variety of such causes thereof. For instance, the bacterial ARI classifier is obtained from a cohort having bacterial infections from multiple bacterial genera and/or species, the viral ARI classifier is obtained from a cohort having viral infections from multiple viral genera and/or species, and the non-infectious illness classifier is obtained from a cohort having a non-infectious illness due to multiple non-infectious causes. See, e.g., Table 8. In this way, the respective classifiers obtained are agnostic to the underlying bacteria, virus, and non-infectious cause. In some embodiments, some or all of the subjects with non-infectious causes of illness in the cohort have symptoms consistent with a respiratory infection.

In some embodiments, the signatures may be obtained using a supervised statistical approach known as sparse linear classification in which sets of genes are identified by the model according to their ability to separate phenotypes during a training process that uses the selected set of patient samples. The outcomes of training are gene signatures and classification coefficients for the three comparisons. Together the signatures and coefficients provide a classifier or predictor. Training may also be used to establish threshold or cut-off values. Threshold or cut-off values can be adjusted to change test performance, e.g., test sensitivity and specificity. For example, the threshold for bacterial ARI may be intentionally lowered to increase the sensitivity of the test for bacterial infection, if desired.

In some embodiments, the classifier generating comprises iteratively: (i) assigning a weight for each normalized gene expression value, entering the weight and expression value for each gene into a classifier (e.g., a linear regression classifier) equation and determining a score for outcome for each of the plurality of subjects, then (ii) determining the accuracy of classification for each outcome across the plurality of subjects, and then (iii) adjusting the weight until accuracy of classification is optimized. Genes having a non-zero weight are included in the respective classifier.

In some embodiments, the classifier is a linear regression classifier and said generating comprises converting a score of said classifier to a probability using a link function. As known in the art, the link function specifies the link between the target/output of the model (e.g., probability of bacterial infection) and systematic components (in this instance, the combination of explanatory variables that comprise the predictor) of the linear model. It says how the expected value of the response relates to the linear predictor of explanatory variable.

Methods of Classification

The present disclosure further provides methods for determining whether a patient has a respiratory illness due to a bacterial infection, a viral infection, or a non-infectious cause. The method for making this determination relies upon the use of classifiers obtained as taught herein. The methods may include: a) measuring the expression levels of pre-defined sets of genes (i.e., for one or more of the three signatures); b) normalizing gene expression levels for the technology used to make said measurement; c) taking those values and entering them into a bacterial classifier, a viral classifier and/or non-infectious illness classifier (i.e., predictors) that have pre-defined weighting values (coefficients) for each of the genes in each signature; d) comparing the output of the classifiers to pre-defined thresholds, cut-off values, confidence intervals or ranges of values that indicate likelihood of infection; and optionally e) jointly reporting the results of the classifiers.

A simple overview of such methods is provided in FIG. 3. In this representation, each of the three gene signatures is informative of the patient's host response to a different ARI etiology (bacterial or viral) or to an ill, but not infected, state (NI). These signatures are groups of gene transcripts which have consistent and coordinated increased or decreased levels of expression in response to one of three clinical states: bacterial ARI, viral ARI, or a non-infected but ill state. These signatures are derived using carefully adjudicated groups of patient samples with the condition(s) of interest (training 10).

With reference to FIG. 3, after obtaining a biological sample from the patient (e.g., a blood sample), in some embodiments the mRNA is extracted. The mRNA (or a defined region of each mRNA), is quantified for all, or a subset, of the genes in the signatures. Depending upon the apparatus that is used for quantification, the mRNA may have to be first purified from the sample.

The signature is reflective of a clinical state and is defined relative to at least one of the other two possibilities. For example, the bacterial ARI signature is identified as a group of biomarkers (here, represented by gene mRNA transcripts) that distinguish patients with bacterial ARI and those without bacterial ARI (including patients with viral ARI or non-infectious illness as it pertains to this application). The viral ARI signature is defined by a group of biomarkers that distinguish patients with viral ARI from those without viral ARI (including patients with either bacterial ARI or non-infectious illness). The non-infectious illness signature is defined by a group of biomarkers that distinguish patients with non-infectious causes of illness relative to those with either bacterial or viral ARI.

The normalized expression levels of each gene of the signature (e.g., first column Table 9) are the explanatory or independent variables or features used in the classifier. As an example, the classifier may have a general form as a probit regression formulation:

P(having condition)=Φ(β₁ X ₁+β₂ X ₂+ . . . +β_(d) X _(d))   (equation 1)

where the condition is bacterial ARI, viral ARI, or non-infection illness; Φ(⋅) is the probit (or logistic, etc.) link function; {β₁,β₂, . . . , β_(d)} are the coefficients obtained during training (e.g., second, third and fourth columns from Table 9) (coefficients may also be denoted {w₁,w₂, . . . , w_(d)} as “weights” herein); {X₁,X₂, . . . , X_(d)} are the normalized gene expression levels of the signature; and d is the size of the signature (i.e., number of genes).

As would be understood by one skilled in the art, the value of the coefficients for each explanatory variable will change for each technology platform used to measure the expression of the genes or a subset of genes used in the probit regression model. For example, for gene expression measured by Affymetrix U133A 2.0 microarray, the coefficients for each of the features in the classifier algorithm are shown in Table 9.

The sensitivity, specificity, and overall accuracy of each classifier may be optimized by changing the threshold for classification using receiving operating characteristic (ROC) curves.

Another aspect of the present disclosure provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of a) obtaining a biological sample from the subject; b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes (i.e., three signatures); c) normalizing gene expression levels for the technology used to make said measurement to generate a normalized value; d) entering the normalized value into a bacterial classifier, a viral classifier and non-infectious illness classifier (i.e., predictors) that have pre-defined weighting values (coefficients) for each of the genes in each signature; e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; and e) classifying the sample as being of bacterial etiology, viral etiology, or noninfectious illness. In some embodiments, the method further comprises generating a report assigning the patient a score indicating the probability of the etiology of the ARI.

The classifiers that are developed during training and using a training set of samples are applied for prediction purposes to diagnose new individuals (“classification”). For each subject or patient, a biological sample is taken and the normalized levels of expression (i.e., the relative amount of mRNA expression) in the sample of each of the genes specified by the signatures found during training are the input for the classifiers. The classifiers also use the weighting coefficients discovered during training for each gene. As outputs, the classifiers are used to compute three probability values. Each probability value may be used to determine the likelihood of the three considered clinical states: bacterial ARI, viral ARI, and non-infectious illness.

In some embodiments, the results of each of the classifiers—the probability a new subject or patient has a bacterial ARI, viral ARI, or non-infectious illness—are reported. In final form, the three signatures with their corresponding coefficients are applied to an individual patient to obtain three probability values, namely probability of having a bacterial ARI, viral ARI, and a non-infectious illness. In some embodiments, these values may be reported relative to a reference range that indicates the confidence with which the classification is made. In some embodiments, the output of the classifier may be compared to a threshold value, for example, to report a “positive” in the case that the classifier score or probability exceeds the threshold indicating the presence of one or more of a bacterial ARI, viral ARI, or non-infectious illness. If the classifier score or probability fails to reach the threshold, the result would be reported as “negative” for the respective condition. Optionally, the values for bacterial and viral ARI alone are reported and the report is silent on the likelihood of ill but not infected.

It should be noted that a classifier obtained with one platform may not show optimal performance on another platform. This could be due to the promiscuity of probes or other technical issues particular to the platform. Accordingly, also described herein are methods to adapt a signature as taught herein from one platform for another.

For example, a signature obtained from an Affymetrix platform may be adapted to a TLDA platform by the use of corresponding TLDA probes for the genes in the signature and/or substitute genes correlated with those in the signature, for the Affymetrix platform. Table 1 shows a list of Affymetrix probes and the genes they measure, plus “replacement genes” that are introduced as replacements for gene probes that either may not perform well on the TLDA platform for technical reasons or to replace those Affymetrix probes for which there is no cognate TLDA probe. These replacements may indicate highly correlated genes or may be probes that bind to a different location in the same gene transcript. Additional genes may be included, such as pan-viral gene probes. The weights shown in Table 1 are weights calculated for a classifier implemented on the microarray platform. Weights that have not been estimated are indicated by “NA” in the table. (Example 4 below provides the completed translation of these classifiers to the TLDA platform.) Reference probes for TLDA (i.e., normalization genes, e.g., TRAP1, PPIB, GAPDH and 18S) also have “NA” in the columns for weights and Affymetrix probeset ID (these are not part of the classifier). Additional gene probes that do not necessarily correspond to the Affymetrix probeset also have “NA” in the Affymetrix probeset ID column.

TABLE 1 Preliminary Gene List for TLDA platform Columns are as follows: Column 1: Affymetrix probeset ID - this was the probeset identified in the Affy discovery analyses (primary probeset) Columns 2, 3, 4: estimated coefficients (weights) for contribution of each probates to the 3 classifiers from Affymetrix weights Column 5: Gene name AFFXProbeSet Bacterial Viral NI Gene 216867_s_at 0.0534745 0 0 PDGFA 203313_s_at 1.09463 0 0 TGIF1 NA NA NA NA TRAP1 NA NA NA NA PPIB 202720_at 0 0.0787402 0 TES 210657_s_at NA NA NA SEPT4 NA NA NA NA EPHB3 NA NA NA NA SYDE1 202864_s_at 0 0.100019 0 SP100 213633_at 1.01336 0 0 SH3BP1 NA NA NA NA 18S NA NA NA NA 18S NA NA NA NA GIT2 205153_s_at 0.132886 0 0 CD40 202709_at 0.427849 0 0 FMOD 202973_x_at 0.112081 0 0 FAM13A 204415_at NA NA NA IFI6 202509_s_at 0 0 0.416714 TNFAIP2 200042_at 0 0.0389975 0 RTCB 206371_at 0.0439022 0 0 FOLR3 212914_at 0 0 0.0099678 CBX7 215804_at 1.94364 0 0 EPHA1 215268_at 0.0381782 0 0 KIAA0754 203153_at NA NA NA IFIT1 217502_at NA NA NA IFIT2 205569_at NA NA NA LAMP3 218943_s_at NA NA NA DDX58 NA NA NA NA GAPDH 213300_at 0.578303 0 0 ATG2A 200663_at 0.176027 0 0 CD63 216303_s_at 0.31126 0 0 MTMR1 NA NA NA NA ICAM2 NA NA NA NA EXOSC4 208702_x_at 0 0 0.0426262 APLP2 NA NA NA NA 18S NA NA NA NA 18S NA NA NA NA FPGS 217408_at 0 1.089 0.0690681 MRPS18B 206918_s_at 1.00926 0 0 CPNE1 208029_s_at 0.020511 0 0.394049 LAPTM4B 203153_at 0.133743 0 0 IFIT1 NA NA NA NA DECR1 200986_at NA NA NA SERPING1 214097_at 0.211804 0.576801 0 RPS21 204392_at 0 0.129465 0 CAMK1 219382_at 0.866643 0 0 SERTAD3 205048_s_at 0.0114514 0 0 PSPH 205552_s_at NA NA NA OAS1 219684_at NA NA NA RTP4 221491_x_at 0.651431 0 0 HLA-DRB3 NA NA NA NA TRAP1 NA NA NA NA PPIB 216571_at 0.878426 0 0 SMPD1 215606_s_at 0.479765 0 0 ERC1 44673_at 0.0307987 0 0 SIGLEC1 222059_at 0 0.112261 0 ZNF335 NA NA NA NA MRC2 209031_at 0 0 0.237916 CADM1 209919_x_at 0.613197 0 0 GGT1 214085_x_at 0.367611 0 0 GLIPR1 NA NA NA NA ELF4 200947_s_at 1.78944 0 0 GLUD1 206676_at 0 0 0.0774651 CEACAM8 NA NA NA NA IFNGR2 207718_x_at 0.0392962 0 0 CYP2A7 220308_at 0 0.0345586 0 CCDC19 205200_at 0.87833 0 0 CLEC3B 202284_s_at 0.356457 0 0 CDKN1A 213223_at 0.686657 0 0 RPL28 205312_at 0 0 0.394304 SPI1 212035_s_at 2.0241 0 1.3618 EXOC7 218306_s_at 0 0 0.784894 HERC1 205008_s_at 0 0.223868 0 CIB2 219777_at 0 0.25509 0 GIMAP6 218812_s_at 0.967987 0 0 ORAI2 NA NA NA NA GAPDH 208736_at 0 0.582264 0.0862941 ARPC3 203455_s_at 0 0 0.0805395 SAT1 208545_x_at 0.265408 0 0 TAF4 NA NA NA NA TLDC1 202509_s_at NA NA NA TNFAIP2 205098_at 0.116414 0 0 CCR1 222154_s_at NA NA NA SPATS2L 201188_s_at 0.606326 0 0 ITPR3 NA NA NA NA FPGS 205483_s_at NA NA NA ISG15 205965_at 0.02668 0 0 BATF 220059_at 0.86817 0 0 STAP1 214955_at 0.100645 0 0 TMPRSS6 NA NA NA NA DECR1 218595_s_at 0 0 0.422722 HEATR1 221874_at 0.40581 0 0.017015 KIAA1324 205001_s_at 0 0.067117 0 DDX3Y 219211_at NA NA NA USP18 209605_at 0.499338 0 0 TST 212708_at 0.0325637 0 0 MSL1 203392_s_at 0 0.0139199 0 CTBP1 202688_at 0 0.0050837 0 TNFSF10 NA NA NA NA TRAP1 NA NA NA NA PPIB 203979_at 0.00999102 0 0.301178 CYP27A1 204490_s_at 0.00732794 0 0 CD44 206207_at 0.0852924 0 0 CLC 216289_at 0 0.00074607 0 GPR144 201949_x_at 0 0 0.034093 CAPZB NA NA NA NA EXOG 216473_x_at 0 0.0769736 0 DUX4 212900_at 0.0573273 0 0 SEC24A 204439_at NA NA NA IFI44L 212162_at 0 0.0102331 0 KIDINS220 209511_at 0 0.031194 0 POLR2F 214175_x_at 0 0 0.266628 PDLIM4 219863_at NA NA NA HERC5 206896_s_at 0.482822 0 0 GNG7 208886_at 0.149103 0 0 H1FO 212697_at 0 0 1.02451 FAM134C NA NA NA NA FNBP4 202672_s_at NA NA NA ATF3 201341_at 0.109677 0 0 ENC1 210797_s_at 0 0.188667 0 OASL 206647_at 0.0650386 0 0 HBZ 215848_at 0 0.326241 0 SCAPER 213573_at 0 0 0.50859 KPNB1 NA NA NA NA GAPDH NA NA NA NA POLR1C 214582_at 0 0 0.0377349 PDE3B 218700_s_at 0 0.00086067 0 RAB7L1 203045_at 0.850903 0 0 NINJ1 NA NA NA NA ZER1 206133_at NA NA NA XAF1 213797_at NA NA NA RSAD2 219437_s_at 0 0.405445 0.217428 ANKRD11 NA NA NA NA FPGS 212947_at 0.286979 0 0 SLC9A8 NA NA NA NA SOX4 202145_at 0 0.166043 0 LY6E 213633_at 1.01336 0 0 SH3BP1 NA NA NA NA DECR1 210724_at 0 0 0.482166 EMR3 220122_at 0.399475 0 0 MCTP1 218400_at NA NA NA OAS3 201659_s_at 0.110991 0 0 ARL1 214326_x_at 0.698109 0 0.261075 JUND NA NA NA NA MRPS31 217717_s_at 0.638943 0 0 YWHAB 218095_s_at 0.00541128 0.613773 0 TMEM165 NA NA NA NA TRAP1 NA NA NA NA PPIB 219066_at 0 0.221446 0 PPCDC 214022_s_at 0 0 0.0380438 IFITM1 214453_s_at NA NA NA IFI44 215342_s_at 0.0497241 0 0 RABGAP1L 204545_at 0.342478 0 0 PEX6 220935_s_at 0.170358 0 0 CDK5RAP2 201802_at 0.00859629 0 0 SLC29A1 202086_at NA NA NA MX1 209360_s_at 0.319632 0 0 RUNX1 NA NA NA NA LY75-CD302 203275_at 0 0.118256 0 IRF2 NA NA NA NA MYL10 203882_at 0 0.0776936 0 IRF9 206934_at 0.151959 0 0 SIRPB1 207860_at 0.376517 0 0 NCR1 207194_s_at 0.3162 0 0 ICAM4 209396_s_at 0 0 0.0355749 CHI3L1 204750_s_at 0.537475 0 0 DSC2 207840_at 0 0.118889 0 CD160 202411_at 0.0522361 0 0 IFI27 215184_at 0 0.0650331 0 DAPK2 202005_at 0.680527 0 0 ST14 214800_x_at 0 0.103261 0 BTF3 NA NA NA NA GAPDH 207075_at 0.0627344 0 0 NLRP3 206026_s_at NA NA NA TNFAIP6 219523_s_at 0 0 0.07715 TENM3 217593_at 0.0747507 0 0 ZSCAN18 204747_at NA NA NA IFIT3 212657_s_at 0 0 0.254507 IL1RN 204972_at NA NA NA OAS2 207606_s_at 0.299775 0 0 ARHGAP12 NA NA NA NA FPGS 205033_s_at 0 0.0878603 0 DEFA3 219143_s_at 0.415444 0 0 RPP25 208601_s_at 0.270581 0 0 TUBB1 216713_at 0.510039 0 0 KRIT1 NA NA NA NA DECR1 214617_at 0.261957 0 0 PRF1 201055_s_at 0 0 1.25363 HNRNPAO 219055_at 0.0852367 0 0 SRBD1 219130_at 0 0.150771 0 TRMT13 202644_s_at 0.340624 0 0 TNFAIP3 205164_at 0.46638 0 0 GCAT

Further discussion of this example signature for a TLDA platform is provided below in Examples 3 and 4.

This method of determining the etiology of an ARI may be combined with other tests. For example, if the patient is determined to have a viral ARI, a follow-up test may be to determine if influenza A or B can be directly detected or if a host response indicative of such an infection can be detected. Similarly, a follow-up test to a result of bacterial ARI may be to determine if a Gram positive or a Gram negative bacterium can be directly detected or if a host response indicative of such an infection can be detected. In some embodiments, simultaneous testing may be performed to determine the class of infection using the classifiers, and also to test for specific pathogens using pathogen-specific probes or detection methods. See, e.g., US 2015/0284780 to Eley et al. (method for detecting active tuberculosis); US 2014/0323391 to Tsalik et al. (method for classification of bacterial infection).

Methods of Determining a Secondary Classification of an ARI in a Subject

The present disclosure also provides methods of classifying a subject using a secondary classification scheme. Accordingly, another aspect of the present invention provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes (i.e., three signatures); (c) normalizing gene expression levels as required for the technology used to make said measurement to generate a normalized value; (d) entering the normalized value into classifiers (i.e., predictors) that have pre-defined weighting values (coefficients) for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) if the sample is negative for bacteria, repeating step (d) using only the viral classifier and non-infectious classifier; and (g) classifying the sample as being of viral etiology or non-infectious illness.

Another aspect of the present provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes (i.e., three signatures); (c) normalizing gene expression levels for the technology used to make said measurement to generate a normalized value; (d) entering the normalized value into classifiers (i.e., predictors) that have pre-defined weighting values (coefficients) for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) if the sample is negative for virus, repeating step (d) using only the bacteria classifier and non-infectious classifier; and (g) classifying the sample as being of bacterial etiology or noninfectious illness.

Yet another aspect of the present provides a method for determining whether an acute respiratory infection (ARI) in a subject is bacterial in origin, viral in origin, or non-infectious in origin comprising, consisting of, or consisting essentially of (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes (i.e., three signatures); (c) normalizing gene expression levels for the technology used to make said measurement to generate a normalized value; (d) entering the normalized value into classifiers (i.e., predictors) that have pre-defined weighting values (coefficients) for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) if the sample is negative for non-infectious illness, repeating step (d) using only the viral classifier and bacterial classifier; and (g) classifying the sample as being of viral etiology or bacterial etiology.

In some embodiments, the method further comprises generating a report assigning the patient a score indicating the probability of the etiology of the ARI.

Classifying the status of a patient using a secondary classification scheme is shown in FIG. 4. In this example, the bacterial ARI classifier will distinguish between patients with a bacterial ARI from those without a bacterial ARI, which could, instead, be a viral ARI or a non-infectious cause of illness. A secondary classification can then be imposed on those patients with non-bacterial ARI to further discriminate between viral ARI and non-infectious illness. This same process of primary and secondary classification can also be applied to the viral ARI classifier where patients determined not to have a viral infection would then be secondarily classified as having a bacterial ARI or non-infectious cause of illness. Likewise, applying the non-infectious illness classifier as a primary test will determine whether patients have such a non-infectious illness or instead have an infectious cause of symptoms. The secondary classification step would determine if that infectious is due to bacterial or viral pathogens.

Results from the three primary and three secondary classifications can be summed through various techniques by those skilled in the art (such as summation, counts, or average) to produce an actionable report for the provider. In some embodiments, the genes used for this secondary level of classification can be some or all of those presented in Table 2.

In such examples, the three classifiers described above (bacteria classifier, virus classifier and non-infectious illness classifier) are used to perform the 1^(st) level classification. Then for those patients with non-bacterial infection, a secondary classifier is defined to distinguish viral ARI from those with non-infectious illness (FIG. 4, left panel). Similarly, for those patients with non-viral infection, a new classifier is used to distinguish viral from non-infectious illness (FIG. 4, middle panel), and for those patients who are not classified as having a non-infectious illness in the first step, a new classifier is used to distinguish between viral and bacterial ARI (FIG. 4, right panel).

In this two-tier method, nine probabilities may be generated, and those probabilities may be combined in a number of ways. Two strategies are described here as a way to reconcile the three sets of predictions, where each has a probability of bacterial ARI, viral ARI, and non-infectious illness. For example: Highest predicted average probability: All predicted probabilities for bacterial ARI are averaged, as are all the predicted probabilities of viral ARI and, similarly, all predicted probabilities of non-infectious illness. The greatest averaged probability denotes the diagnosis.

Greatest number of predictions: Instead of averaging the predicted probabilities of each condition, the number of times a particular diagnosis is predicted for that patient sample (i.e., bacterial ARI, viral ARI or non-infectious illness) is counted. The best-case scenario is when the three classification schemes give the same answer (e.g., bacterial ARI for scheme 1, bacterial ARI for scheme 2, and bacterial ARI for scheme 3). The worst case is that each scheme nominates a different diagnosis, resulting in a 3-way tie.

Using the training set of patient samples previously described, the Result of Tier 1 classification could be, for example (clinical classification presented in rows; diagnostic test prediction presented in columns) similar to that presented in Table 3.

TABLE 3 bacterial viral ni counts bacterial 82.8 12.8 4.2 58 9 3 viral 3.4 90.4 6.0 4 104 7 ni 9.0 4.5 86.3 8 4 76

Following Tier 2 classification using the highest predicted average probability strategy (clinical classification presented in rows; diagnostic test prediction presented in columns), results may be similar to Table 4.

TABLE 4 Mean (average predictions than max): bacterial viral ni counts bacterial 82.8 11.4 5.7 58 8 4 viral 1.7 91.3 6.9 2 105 8 ni 7.9 7.9 84.0 7 7 74

Following Tier 2 classification using the greatest number of predictions strategy (clinical classification presented in rows; diagnostic test prediction presented in columns), results may be similar to Table 5.

TABLE 5 Max (max predictions then count votes, 7 ties): bacterial viral ni counts bacterial 84.2 11.4 4.2 59 8 3 viral 4.3 89.5 6.0 5 103 7 ni 11.3 7.9 80.6 10 7 71

Classification can be achieved, for example, as described above, and/or as summarized in Table 2. Table 2 summarizes the gene membership in three distinct classification strategies that solve different diagnostic questions. There are a total of 270 probes that collectively comprise three complex classifiers. The first is referred to as BVS (Bacterial ARI, Viral ARI, SIRS), which is the same as that presented below in Example 1. These probes are the same as those presented in Table 9, which offers probe/gene weights used in classification. They also correspond to the genes presented in Table 10.

The second is referred to as 2L for 2-layer or 2-tier. This is the hierarchical scheme presented in FIG. 4.

The third is a one-tier classification scheme, BVSH, which is similar to BVS but also includes a population of healthy controls (similarly described in Example 1). This group has been shown to be a poor control for non-infection, but there are use cases in which discrimination from healthy may be clinically important. For example, this can include the serial measurement of signatures to correlate with convalescence. It may also be used to discriminate patients who have been exposed to an infectious agent and are presymptomatic vs. asymptomatic. In the BVSH scheme, four groups are represented in the training cohort—those with bacterial ARI, viral ARI, SIRS (non-infectious illness), and Healthy. These four groups are used to generate four distinct signatures that distinguish each class from all other possibilities.

Table 2 Legend:

-   Probe=Affymetrix probe ID -   BVS=Three-classifier model trained on patients with Bacterial ARI,     Viral ARI, and Non-Infectious Illness (with respiratory symptoms). 1     denotes this probe is included in this three-classifier model. 0     denotes the probe is not present in this classification scheme. -   BVS-BO=Genes or probes included in the Bacterial ARI classifier as     part of the BVS classification scheme. This classifier specifically     discriminates patients with bacterial ARI from other etiologies     (viral ARI or or 10) -   BVS-VO=As for BVS-BO except this column identifies genes included in     the Viral ARI classifier. This classifier specifically discriminates     patients with viral ARI from other etiologies (bacterial ARI or     non-infectious illness) -   BVS-SO=As for BVS-BO or BVS-VO, except this column identifies genes     included in the non-infectious illness classifier. This classifier     specifically discriminates patients with non-infectious illness from     other etiologies (bacterial or viral ARI) -   2L refers to the two-tier hierarchical classification scheme. A 1 in     this column indicates the specified probe or gene was included in     the classification task. This 2-tier classification scheme is itself     comprised of three separate tiered tasks. The first applies a one     vs. others, where one can be Bacterial ARI, Viral ARI, or     non-infectious illness. If a given subject falls into the “other”     category, a 2^(nd) tier classification occurs that distinguishes     between the remaining possibilities. -   2L-SO is the 1^(st) tier for a model that determines with a given     subject has a non-infectious illness or not, followed by SL-BV which     discriminates between bacterial and viral ARI as possibilities. A 1     in these columns indicates that gene or probe are included in that     specified classification model. 2L-BO and 2L-VS make another 2-tier     classification scheme. 2L-VO and 2L-SB comprise the 3^(rd) model in     the 2-tier classification scheme.

Finally, BVSH refers to a one-level classification scheme that includes healthy individuals in the training cohort and therefore includes a classifier for the healthy state as compared to bacterial ARI, viral ARI, or non-infectious illness. The dark grey BVSH column identifies any gene or probe included in this classification scheme. This scheme is itself comprised by BVSH-BO, BVSH-VO, BVSH-SO, and BVSH-HO with their respective probe/gene compositions denoted by ‘1’ in these columns.

Table 2 provides a summary of use of members of the gene sets for viral, bacterial, and non-infectious illness classifiers that are constructed according to the required task. A ‘1’ indicates membership of the gene in the classifier.

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1 0 0 1 213700_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 213831_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 213907_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 214085_x_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 214097_at 1 1 1 0 1 0 1 1 1 1 0 1 1 0 0 214175_x_at 1 0 0 1 1 1 0 0 0 0 0 0 0 0 0 214321_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 214326_x_at 1 1 0 1 1 1 1 1 0 0 1 1 1 0 1 214511_x_at 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 214582_at 1 0 0 1 1 1 0 0 0 0 0 0 0 0 0 214617_at 1 1 0 0 1 0 0 1 0 0 0 1 1 0 0 214800_x_at 1 0 1 0 1 0 0 0 0 1 0 1 0 1 0 214955_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 215012_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 215088_s_at 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 215184_at 1 0 1 0 1 0 0 0 1 1 0 0 0 0 0 215268_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 215606_s_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 215630_at 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 215696_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 215804_at 1 1 0 0 1 0 1 1 0 0 0 0 0 0 0 215848_at 1 0 1 0 1 0 0 0 1 1 0 1 0 1 1 216289_at 1 0 1 0 1 0 1 0 0 1 0 0 0 0 0 216303_s_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 216473_x_at 1 0 1 0 1 0 0 0 1 1 0 0 0 0 0 216571_at 1 1 0 0 1 0 1 1 0 0 0 0 0 0 0 216676_x_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 216713_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 216748_at 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 216867_s_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 216950_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 217143_s_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 217408_at 1 0 1 1 1 1 0 0 1 1 0 1 0 1 1 217497_at 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 217593_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 217717_s_at 1 1 0 0 1 0 1 1 0 0 0 0 0 0 0 218010_x_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 218040_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 218060_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 218095_s_at 1 0 1 0 1 0 0 0 1 1 0 0 0 0 0 218135_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 218306_s_at 1 0 0 1 1 1 0 0 0 0 1 0 0 0 0 218510_x_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 218523_at 0 0 0 0 1 0 1 0 0 0 0 1 0 0 1 218595_s_at 1 0 0 1 1 1 0 0 0 0 0 1 0 0 1 218637_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 218700_s_at 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 218812_s_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 218818_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 218946_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 218999_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 219055_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 219066_at 1 0 1 0 1 0 0 0 0 1 0 1 0 1 0 219124_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 219130_at 1 0 1 0 1 0 0 0 0 1 0 0 0 0 0 219143_s_at 0 0 0 0 1 0 1 0 0 0 0 1 1 1 0 219269_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 219382_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 219437_s_at 1 0 1 1 1 1 0 0 1 1 0 1 0 1 0 219523_s_at 1 0 0 1 1 1 0 0 0 0 1 0 0 0 0 219577_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 219599_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 219629_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 219669_at 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 219693_at 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 219745_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 219762_s_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 219763_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 219777_at 1 0 1 0 1 0 0 0 0 1 0 0 0 0 0 219872_at 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 219966_x_at 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 219999_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 220036_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 220059_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 220122_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 220308_at 1 0 1 0 1 0 0 0 1 1 0 0 0 0 0 220319_s_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 220646_s_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 220765_s_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 220935_s_at 0 0 0 0 1 0 0 0 0 0 1 1 1 0 0 221032_s_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 221142_s_at 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 221211_s_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 221491_x_at 1 1 0 0 1 0 1 1 0 0 0 1 1 0 0 221874_at 1 1 0 1 1 1 1 1 0 0 0 1 1 0 0 221964_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 222059_at 1 0 1 0 1 0 0 0 0 1 0 0 0 0 0 222186_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 222297_x_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 222330_at 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 320_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 44673_at 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 49329_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 49452_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 215185_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 AFFX- 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 HUMGAPDH/ M33197_M_at 206512_at 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 211781_x_at 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 216635_at 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 216943_at 1 1 0 0 1 0 1 1 0 0 0 0 0 0 0 217079_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 220352_x_at 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 Affymetrix BVSH- Gene Probe ID HO Symbol RefSeq ID Gene Name 200042_at 0 HSPC117 NM_014306 chromosome 22 open reading frame 28 200073_s_at 1 HNRPD NM_031369; heterogeneous nuclear ribonucleoprotein D (AU-rich NM_001003810; element RNA binding protein 1, 37 kDa) NM_031370; NM_002138 200602_at 1 APP NM_000484; amyloid beta (A4) precursor protein NM_201414; NM_001136131; NM_201413; NM_001136130; NM_001136016; NM_001136129 200663_at 0 CD63 NM_001780; CD63 molecule NM_001040034 200709_at 0 FKBP1A NM_000801; FK506 binding protein 1A, 12 kDa NM_054014 200947_s_at 0 GLUD1 NM_005271 glutamate dehydrogenase 1 201055_s_at 0 HNRPA0 NM_006805 heterogeneous nuclear ribonucleoprotein A0 201162_at 1 IGFBP7 NM_001553 insulin-like growth factor binding protein 7 201166_s_at 1 PUM1 NM_014676; pumilio homolog 1 (Drosophila) NM_001020658 201188_s_at 0 ITPR3 NM_002224 inositol 1,4,5-triphosphate receptor, type 3 201341_at 0 ENC1 NM_003633 ectodermal-neural cortex (with BTB-like domain) 201369_s_at 0 ZFP36L2 NM_006887 zinc finger protein 36, C3H type-like 2 201392_s_at 0 IGF2R NM_000876 insulin-like growth factor 2 receptor 201454_s_at 0 NPEPPS NM_006310; hypothetical protein FLJ11822; aminopeptidase XM_001725441; puromycin sensitive XM_001725426 201464_x_at 0 JUN NM_002228 jun oncogene 201601_x_at 1 IFITM1 NM_003641 interferon induced transmembrane protein 1 (9-27) 201651_s_at 1 PACSIN2 NM_007229 protein kinase C and casein kinase substrate in neurons 2 201659_s_at 0 ARL1 NM_001177 ADP-ribosylation factor-like 1 201802_at 0 SLC29A1 NM_001078176; solute carrier family 29 (nucleoside transporters), NM_001078177; member 1 NM_001078175; NM_004955; NM_001078174 201890_at 0 RRM2 NM_001034; ribonucleotide reductase M2 polypeptide NM_001165931 201949_x_at 0 CAPZB NM_004930 capping protein (actin filament) muscle Z-line, beta 201952_at 0 ALCAM XM_001720217; hypothetical protein LOC100133690; activated NM_001627 leukocyte cell adhesion molecule 201972_at 1 ATP6V1A NM_001690 ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A 201992_s_at 0 KIF5B NM_004521 kinesin family member 5B 202005_at 0 ST14 NM_021978 suppression of tumorigenicity 14 (colon carcinoma) 202083_s_at 1 SEC14L1 NM_001143998; SEC14-like 1 (S. cerevisiae); SEC14-like 1 pseudogene NM_001039573; NM_001144001; NM_001143999; NM_003003 202090_s_at 0 UQCR NM_006830 ubiquinol-cytochrome c reductase, 6.4 kDa subunit 202145_at 0 LY6E NM_002346; lymphocyte antigen 6 complex, locus E NM_001127213 202160_at 1 CREBBP NM_004380; CREB binding protein NM_001079846 202266_at 0 TTRAP NM_016614 TRAF and TNF receptor associated protein 202284_s_at 0 CDKN1A NM_078467; cyclin-dependent kinase inhibitor 1A (p21, Cip1) NM_000389 202411_at 0 IFI27 NM_005532; interferon, alpha-inducible protein 27 NM_001130080 202505_at 0 SNRPB2 NM_003092; small nuclear ribonucleoprotein polypeptide B″ NM_198220 202509_s_at 0 TNFAIP2 NM_006291 tumor necrosis factor, alpha-induced protein 2 202579_x_at 0 HMGN4 NM_006353 high mobility group nucleosomal binding domain 4 202589_at 0 TYMS NM_001071 thymidylate synthetase 202617_s_at 0 MECP2 NM_001110792; methyl CpG binding protein 2 (Rett syndrome) NM_004992 202644_s_at 0 TNFAIP3 NM_006290 tumor necrosis factor, alpha-induced protein 3 202679_at 0 NPC1 NM_000271 Niemann-Pick disease, type C1 202688_at 0 TNFSF10 NM_003810 tumor necrosis factor (ligand) superfamily, member 10 202709_at 0 FMOD NM_002023 fibromodulin 202720_at 0 TES NM_152829; testis derived transcript (3 LIM domains) NM_015641 202748_at 0 GBP2 NM_004120 guanylate binding protein 2, interferon-inducible 202864_s_at 0 SP100 NM_003113; SP100 nuclear antigen NM_001080391 202973_x_at 0 FAM13A1 NM_014883; family with sequence similarity 13, member A NM_001015045 203023_at 0 HSPC111 NM_016391 NOP16 nucleolar protein homolog (yeast) 203045_at 0 NINJ1 NM_004148 ninjurin 1 203153_at 0 IFIT1 NM_001548 interferon-induced protein with tetratricopeptide repeats 1 203275_at 0 IRF2 NM_002199 interferon regulatory factor 2 203290_at 0 HLA-DQA1 NM_002122; similar to hCG2042724; similar to HLA class II XM_001719804; histocompatibility antigen, DQ(1) alpha chain XM_001129369; precursor (DC-4 alpha chain); major XM_001722105 histocompatibility complex, class II, DQ alpha 1 203313_s_at 0 TGIF NM_173211; TGFB-induced factor homeobox 1 NM_173210; NM_003244; NM_174886; NM_173209; NM_173208; NM_173207; NM_170695 203392_s_at 0 CTBP1 NM_001328; C-terminal binding protein 1 NM_001012614 203414_at 1 MMD NM_012329 monocyte to macrophage differentiation-associated 203455_s_at 0 SAT NM_002970 spermidine/spermine N1-acetyltransferase 1 203570_at 0 LOXL1 NM_005576 lysyl oxidase-like 1 203615_x_at 0 SULT1A1 NM_177529; sulfotransferase family, cytosolic, 1A, phenol- NM_177530; preferring, member 1 NM_177534; NM_001055; NM_177536 203633_at 1 CPT1A NM_001876; carnitine palmitoyltransferase 1A (liver) NM_001031847 203717_at 1 DPP4 NM_001935 dipeptidyl-peptidase 4 203882_at 0 ISGF3G NM_006084 interferon regulatory factor 9 203940_s_at 0 VASH1 NM_014909 vasohibin 1 203979_at 0 CYP27A1 NM_000784 cytochrome P450, family 27, subfamily A, polypeptide 1 204069_at 1 MEIS1 NM_002398 Meis homeobox 1 204392_at 0 CAMK1 NM_003656 calcium/calmodulin-dependent protein kinase I 204490_s_at 0 CD44 NM_000610; CD44 molecule (Indian blood group) NM_001001389; NM_001001390; NM_001001391; NM_001001392 204545_at 0 PEX6 NM_000287 peroxisomal biogenesis factor 6 204592_at 1 DLG4 NM_001365; discs, large homolog 4 (Drosophila) NM_001128827 204647_at 0 HOMER3 NM_001145724; homer homolog 3 (Drosophila) NM_004838; NM_001145722; NM_001145721 204724_s_at 0 COL9A3 NM_001853 collagen, type IX, alpha 3 204750_s_at 0 DSC2 NM_004949; desmocollin 2 NM_024422 204853_at 0 ORC2L NM_006190 origin recognition complex, subunit 2-like (yeast) 204858_s_at 1 ECGF1 NM_001953; thymidine phosphorylase NM_001113755; NM_001113756 204981_at 0 SLC22A18 NM_002555; solute carrier family 22, member 18 NM_183233 205001_s_at 0 DDX3Y NM_001122665; DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, γ-linked NM_004660 205008_s_at 0 CIB2 NM_006383 calcium and integrin binding family member 2 205033_s_at 0 DEFA1 /// NM_004084; defensin, alpha 1 DEFA3 NM_001042500 205048_s_at 0 PSPH NM_004577 phosphoserine phosphatase-like; phosphoserine phosphatase 205053_at 0 PRIM1 NM_000946 primase, DNA, polypeptide 1 (49 kDa) 205098_at 0 CCR1 NM_001295 chemokine (C-C motif) receptor 1 205153_s_at 0 CD40 NM_152854; CD40 molecule, TNF receptor superfamily member 5 NM_001250 205164_at 0 GCAT NM_014291; glycine C-acetyltransferase (2-amino-3-ketobutyrate NM_001171690 coenzyme A ligase) 205200_at 0 CLEC3B NM_003278 C-type lectin domain family 3, member B 205312_at 0 SPI1 NM_001080547; spleen focus forming virus (SFFV) proviral NM_003120 integration oncogene spi1 205376_at 0 INPP4B NM_003866; inositol polyphosphate-4-phosphatase, type II, NM_001101669 105 kDa 205382_s_at 0 DF NM_001928 complement factor D (adipsin) 205826_at 0 MYOM2 NM_003970 myomesin (M-protein) 2, 165 kDa 206005_s_at 1 C6orf84 NM_014895 KIAA1009 206035_at 0 REL NM_002908 v-rel reticuloendotheliosis viral oncogene homolog (avian) 206082_at 0 NM_006674 HLA complex P5 206207_at 0 CLC NM_001828 Charcot-Leyden crystal protein 206214_at 0 PLA2G7 NM_005084; phospholipase A2, group VII (platelet-activating NM_001168357 factor acetylhydrolase, plasma) 206371_at 0 FOLR3 NM_000804 folate receptor 3 (gamma) 206508_at 1 TNFSF7 NM_001252 CD70 molecule 206558_at 0 SIM2 NM_009586; single-minded homolog 2 (Drosophila) NM_005069 206647_at 0 HBZ NM_005332 hemoglobin, zeta 206676_at 0 CEACAM8 NM_001816 carcinoembryonic antigen-related cell adhesion molecule 8 206734_at 0 JRKL NM_003772 jerky homolog-like (mouse) 206896_s_at 0 GNG7 NM_052847 guanine nucleotide binding protein (G protein), gamma 7 206918_s_at 0 CPNE1 NM_152929; RNA binding motif protein 12; copine I NM_152928; NM_152927; NM_003915; NM_152931; NM_152930; NM_006047; NM_152925; NM_152926; NM_152838 206934_at 0 SIRPB1 NM_001135844; signal-regulatory protein beta 1 NM_006065; NM_001083910 207008_at 0 IL8RB NM_001168298; interleukin 8 receptor, beta NM_001557 207075_at 0 CIAS1 NM_004895; NLR family, pyrin domain containing 3 NM_001079821; NM_001127462; NM_001127461; NM_183395 207194_s_at 0 ICAM4 NM_022377; intercellular adhesion molecule 4 (Landsteiner- NM_001544; Wiener blood group) NM_001039132 207244_x_at 0 CYP2A6 NM_000762 cytochrome P450, family 2, subfamily A, polypeptide 6 207306_at 0 TCF15 NM_004609 transcription factor 15 (basic helix-loop-helix) 207436_x_at 0 KIAA0894 ambiguous (pending) 207536_s_at 0 TNFRSF9 NM_001561 tumor necrosis factor receptor superfamily, member 9 207606_s_at 0 ARHGAP12 NM_018287 Rho GTPase activating protein 12 207718_x_at 0 CYP2A6 /// NM_000764; cytochrome P450, family 2, subfamily A, polypeptide 7 CYP2A7 /// NM_030589 CYP2A7P1 /// CYP2A13 207721_x_at 0 HINT1 NM_005340 histidine triad nucleotide binding protein 1 207808_s_at 0 PROS1 NM_000313 protein S (alpha) 207840_at 0 CD160 NM_007053 CD160 molecule 207860_at 0 NCR1 NM_001145457; natural cytotoxicity triggering receptor 1 NM_001145458; NM_004829 207983_s_at 1 STAG2 NM_006603; stromal antigen 2 NM_001042749; NM_001042751; NM_001042750 208029_s_at 0 LAPTM4B NM_018407 lysosomal protein transmembrane 4 beta 208241_at 0 NRG1 NM_001160001; neuregulin 1 NM_001159995; NM_001160007; NM_001160008; NM_001159996; NM_001159999; NM_001160002; NM_001160004; NM_004495; NM_001160005; NM_013964; NM_013960; NM_013962; NM_013961; NM_013959; NM_013958; NM_013957; NM_013956 208501_at 0 GFI1B NM_001135031, growth factor independent 1B transcription NM_004188 repressor 208545_x_at 0 TAF4 NM_003185 TAF4 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 135 kDa 208601_s_at 0 TUBB1 NM_030773 tubulin, beta 1 208702_x_at 0 APLP2 NM_001642; amyloid beta (A4) precursor-like protein 2 NM_001142277; NM_001142278; NM_001142276 208710_s_at 0 AP3D1 NM_003938; adaptor-related protein complex 3, delta 1 subunit NM_001077523 208736_at 0 ARPC3 NM_005719 similar to actin related protein ⅔ complex subunit 3; hypothetical LOC729841; actin related protein 2/3 complex, subunit 3, 21 kDa 208743_s_at 0 YWHAB NM_139323; tyrosine 3-monooxygenase/tryptophan 5- NM_003404 monooxygenase activation protein, beta polypeptide 208782_at 0 FSTL1 NM_007085 follistatin-like 1 208886_at 0 H1F0 NM_005318 H1 histone family, member 0 208974_x_at 0 KPNB1 NM_002265 karyopherin (importin) beta 1 209031_at 0 IGSF4 NM_014333; cell adhesion molecule 1 NM_001098517 209218_at 0 SQLE NM_003129 squalene epoxidase 209360_s_at 0 RUNX1 NM_001122607; runt-related transcription factor 1 NM_001001890; NM_001754 209396_s_at 0 CHI3L1 NM_001276 chitinase 3-like 1 (cartilage glycoprotein-39) 209422_at 1 PHF20 NM_016436 PHD finger protein 20 209511_at 0 POLR2F NM_021974 polymerase (RNA) II (DNA directed) polypeptide F 209605_at 0 TST NM_003312 thiosulfate sulfurtransferase (rhodanese) 209691_s_at 0 DOK4 NM_018110 docking protein 4 209906_at 0 C3AR1 NM_004054 complement component 3a receptor 1 209919_x_at 0 GGT1 XM_001129425; gamma-glutamyltransferase light chain 3; gamma- NM_013430; glutamyltransferase 4 pseudogene; gamma- NM_001032365; glutamyltransferase 2; gamma-glutamyltransferase NM_005265; 1; gamma-glutamyltransferase light chain 5 NM_001032364; pseudogene XM_001129377 210164_at 0 GZMB NM_004131 granzyme B (granzyme 2, cytotoxic T-lymphocyte- associated serine esterase 1) 210172_at 0 SF1 NM_004630; splicing factor 1 NM_201995; NM_201997; NM_201998 210240_s_at 0 CDKN2D NM_001800; cyclin-dependent kinase inhibitor 2D (p19, inhibits NM_079421 CDK4) 210365_at 0 RUNX1 NM_001122607; runt-related transcription factor 1 NM_001001890; NM_001754 210499_s_at 0 PQBP1 NM_005710; polyglutamine binding protein 1 NM_001032384; NM_001032383; NM_001167989; NM_001167990; NM_144495; NM_001167992; NM_001032381; NM_001032382 210724_at 0 EMR3 NM_032571 egf-like module containing, mucin-like, hormone receptor-like 3 210797_s_at 0 OASL NM_198213; 2′-5′-oligoadenylate synthetase-like NM_003733 210846_x_at 0 TRIM14 NM_033219; tripartite motif-containing 14 NM_033220; NM_014783; NM_033221 211137_s_at 0 ATP2C1 NM_014382; ATPase, Ca++ transporting, type 2C, member 1 NM_001001486; NM_001001487; NM_001001485 211792_s_at 0 CDKN2C NM_001262; cyclin-dependent kinase inhibitor 2C (p18, inhibits NM_078626 CDK4) 211878_at 1 XM_001718220 immunoglobulin heavy constant gamma 1 (G1m marker); immunoglobulin heavy constant mu; immunoglobulin heavy variable 3-7; immunoglobulin heavy constant gamma 3 (G3m marker); immunoglobulin heavy variable 3-11 (gene/pseudogene); immunoglobulin heavy variable 4-31; immunoglobulin heavy locus 211966_at 0 COL4A2 NM_001846 collagen, type IV, alpha 2 212035_s_at 0 EXOC7 NM_001145298; exocyst complex component 7 NM_001145299; NM_015219; NM_001145297; NM_001145296; NM_001013839 212036_s_at 1 PNN NM_002687 pinin, desmosome associated protein 212118_at 0 RFP NM_006510 tripartite motif-containing 27 212162_at 0 KIDINS220 NM_020738 kinase D-interacting substrate, 220 kDa 212574_x_at 0 C19orf6 NM_033420; chromosome 19 open reading frame 6 NM_001033026 212590_at 0 RRAS2 XM_001726427; related RAS viral (r-ras) oncogene homolog 2; similar NM_012250; to related RAS viral (r-ras) oncogene homolog 2 XM_001726471; NM_001102669; XM_001726315 212655_at 1 ZCCHC14 NM_015144 zinc finger, CCHC domain containing 14 212657_s_at 0 IL1RN NM_000577; interleukin 1 receptor antagonist NM_173841; NM_173842; NM_173843 212659_s_at 0 IL1RN NM_000577; interleukin 1 receptor antagonist NM_173841; NM_173842; NM_173843 212676_at 0 NF1 NM_000267; neurofibromin 1 NM_001042492; NM_001128147 212697_at 0 LOC162427 NM_178126 family with sequence similarity 134, member C 212708_at 0 LOC339287 NM_001012241 male-specific lethal 1 homolog (Drosophila) 212810_s_at 0 SLC1A4 NM_003038; solute carrier family 1 (glutamate/neutral amino NM_001135581 acid transporter), member 4 212816_s_at 0 CBS NM_000071 cystathionine-beta-synthase 212914_at 0 CBX7 NM_175709 chromobox homolog 7 212947_at 0 SLC9A8 NM_015266 solute carrier family 9 (sodium/hydrogen exchanger), member 8 213223_at 0 RPL28 NM_001136134; ribosomal protein L28 NM_000991; NM_001136137; NM_001136135; NM_001136136 213300_at 0 KIAA0404 NM_015104 ATG2 autophagy related 2 homolog A (S. cerevisiae) 213422_s_at 0 MXRA8 NM_032348 matrix-remodelling associated 8 213573_at 0 KPNB1 NM_002265 karyopherin (importin) beta 1 213633_at 1 SH3BP1 NM_018957 SH3-domain binding protein 1 213700_s_at 1 PKM2 NM_002654; similar to Pyruvate kinase, isozymes M1/M2 NM_182471; (Pyruvate kinase muscle isozyme) (Cytosolic thyroid NM_182470; hormone-binding protein) (CTHBP) (THBP1); XM_001719890 pyruvate kinase, muscle 213831_at 0 HLA-DOA1 NM_002122; similar to hCG2042724; similar to HLA class II XM_001719804; histocompatibility antigen, DQ(1) alpha chain XM_001129369; precursor (DC-4 alpha chain); major XM_001722105 histocompatibility complex, class II, DQ alpha 1 213907_at 0 EEF1E1 NM_004280; eukaryotic translation elongation factor 1 epsilon 1 NM_001135650 214085_x_at 0 GLIPR1 NM_006851 GLI pathogenesis-related 1 214097_at 0 RPS21 NM_001024 ribosomal protein S21 214175_x_at 0 PDLIM4 NM_003687; PDZ and LIM domain 4 NM_001131027 214321_at 0 NOV NM_002514 nephroblastoma overexpressed gene 214326_x_at 0 JUND NM_005354 jun D proto-oncogene 214511_x_at 0 FCGR1A /// NM_001017986; Fc fragment of IgG, high affinity Ib, receptor (CD64) LOC440607 NM_001004340 214582_at 0 PDE3B NM_000922 phosphodiesterase 3B, cGMP-inhibited 214617_at 0 PRF1 NM_005041; perforin 1 (pore forming protein) NM_001083116 214800_x_at 0 BTF3 /// NM_001037637; basic transcription factor 3; basic transcription LOC345829 NM_001207 factor 3, like 1 pseudogene 214955_at 0 TMPRSS6 NM_153609 transmembrane protease, serine 6 215012_at 0 ZNF451 NM_001031623; zinc finger protein 451 NM_015555 215088_s_at 0 SDHC NM_003001; succinate dehydrogenase complex, subunit C, NM_001035513; integral membrane protein, 15 kDa NM_001035511; NM_001035512 215184_at 0 DAPK2 NM_014326 death-associated protein kinase 2 215268_at 0 KIAA0754 NM_015038 hypothetical LOC643314 215606_s_at 0 RAB6IP2 NM_178040; ELKS/RAB6-interacting/CAST family member 1 NM_015064; NM_178037; NM_178038; NM_178039 215630_at 0 NM_015150 raftlin, lipid raft linker 1 215696_s_at 0 KIAA0310 NM_014866 SEC16 homolog A (S. cerevisiae) 215804_at 0 EPHA1 NM_005232 EPH receptor A1 215848_at 0 ZNF291 NM_001145923; S-phase cyclin A-associated protein in the ER NM_020843 216289_at 0 XM_002347085; G protein-coupled receptor 144 XM_002342934; XM_002346195; NM_001161808 216303_s_at 0 MTMR1 NM_003828 myotubularin related protein 1 216473_x_at 0 DUX4 /// XM_927996; double homeobox, 4-like; similar to double LOC399839 XM_001720078; homeobox 4c; similar to double homeobox, 4; /// XM_001722088; double homeobox, 4 LOC401650 NM_001164467; /// XM_928023; LOC440013 XM_495858; /// XM_941455; LOC440014 NM_001127386; /// XM_001720082; LOC440015 XM_001720798; /// XM_496731; LOC440016 NM_001127387; /// XM_495854; LOC440017 XM_495855; /// NM_001127388; LOC441056 NM_033178; NM_001127389; XM_001724713 216571_at 0 NM_000543; sphingomyelin phosphodiesterase 1, acid lysosomal NM_001007593 216676_x_at 0 KIR3DL3 NM_153443 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 3 216713_at 0 KRIT1 NM_194454; KRIT1, ankyrin repeat containing NM_001013406; NM_004912; NM_194456; NM_194455 216748_at 0 PYHIN1 NM_198928; pyrin and HIN domain family, member 1 NM_152501; NM_198930; NM_198929 216867_s_at 0 PDGFA NM_033023; platelet-derived growth factor alpha polypeptide NM_002607 216950_s_at 1 FCGR1A NM_000566 Fc fragment of IgG, high affinity Ic, receptor (CD64); Fc fragment of IgG, high affinity Ia, receptor (CD64) 217143_s_at 0 TRA@ /// ambiguous (pending) TRD@ 217408_at 0 MRPS18B NM_014046 mitochondrial ribosomal protein S18B 217497_at 0 ECGF1 NM_001953; thymidine phosphorylase NM_001113755; NM_001113756 217593_at 0 ZNF447 NM_001145542; zinc finger and SCAN domain containing 18 NM_001145543; NM_001145544; NM_023926 217717_s_at 0 YWHAB NM_139323; tyrosine 3-monooxygenase/tryptophan 5- NM_003404 monooxygenase activation protein, beta polypeptide 218010_x_at 0 C20orf149 NM_024299 pancreatic progenitor cell differentiation and proliferation factor homolog (zebrafish) 218040_at 0 PRPF38B NM_018061 PRP38 pre-mRNA processing factor 38 (yeast) domain containing B 218060_s_at 1 FLJ13154 NM_024598 chromosome 16 open reading frame 57 218095_s_at 0 TPARL NM_018475 transmembrane protein 165 218135_at 1 PTX1 NM_016570 ERGIC and golgi 2 218306_s_at 0 HERC1 NM_003922 hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 218510_x_at 0 FLJ20152 NM_001034850; family with sequence similarity 134, member B NM_019000 218523_at 0 LHPP NM_022126; phospholysine phosphohistidine inorganic NM_001167880 pyrophosphate phosphatase 218595_s_at 0 HEATR1 NM_018072 HEAT repeat containing 1 218637_at 1 IMPACT NM_018439 Impact homolog (mouse) 218700_s_at 0 RAB7L1 NM_001135664; RAB7, member RAS oncogene family-like 1 NM_001135663; NM_001135662; NM_003929 218812_s_at 0 C7orf19 NM_032831; ORAI calcium release-activated calcium modulator 2 NM_001126340 218818_at 0 FHL3 NM_004468 four and a half LIM domains 3 218946_at 1 HIRIP5 NM_001002755; NFU1 iron-sulfur cluster scaffold homolog (S. cerevisiae) NM_001002756; NM_001002757; NM_015700 218999_at 0 FLJ11000 NM_018295 transmembrane protein 140 219055_at 0 FLJ10379 NM_018079 S1 RNA binding domain 1 219066_at 0 PPCDC NM_021823 phosphopantothenoylcysteine decarboxylase 219124_at 0 C8orf41 NM_001102401; chromosome 8 open reading frame 41 NM_025115 219130_at 0 FLJ10287 NM_019083 coiled-coil domain containing 76 219143_s_at 0 RPP25 NM_017793 ribonuclease P/MRP 25 kDa subunit 219269_at 0 FLJ21616 NM_001135726; homeobox containing 1 NM_024567 219382_at 0 SERTAD3 NM_013368; SERTA domain containing 3 NM_203344 219437_s_at 0 ANKRD11 XM_001720760; ankyrin repeat domain 11; hypothetical protein NM_013275; LOC100128265 XM_001721661; XM_001721649 219523_s_at 0 ODZ3 NM_001080477 odz, odd Oz/ten-m homolog 3 (Drosophila) 219577_s_at 0 ABCA7 NM_019112 ATP-binding cassette, sub-family A (ABC1), member 7 219599_at 0 PRO1843 NM_001417 similar to eukaryotic translation initiation factor 4H; eukaryotic translation initiation factor 4B 219629_at 0 C22orf8 NM_017911; family with sequence similarity 118, member A NM_001104595 219669_at 0 CD177 NM_020406 CD177 molecule 219693_at 0 AGPAT4 NM_020133 1-acylglycerol-3-phosphate O-acyltransferase 4 (lysophosphatidic acid acyltransferase, delta) 219745_at 0 C10orf77 NM_024789 transmembrane protein 180 219762_s_at 0 RPL36 NM_033643; ribosomal protein L36; ribosomal protein L36 NM_015414 pseudogene 14 219763_at 0 DENND1A NM_020946; DENN/MADD domain containing 1A NM_024820 219777_at 0 GIMAP6 NM_024711 GTPase, IMAP family member 6 219872_at 0 DKFZp434L142 NM_001031700; chromosome 4 open reading frame 18 NM_016613; NM_001128424 219966_x_at 1 BANP NM_017869; BTG3 associated nuclear protein NM_079837 219999_at 0 MAN2A2 NM_006122 mannosidase, alpha, class 2A, member 2 220036_s_at 0 LMBR1L NM_018113 limb region 1 homolog (mouse)-like 220059_at 0 BRDG1 NM_012108 signal transducing adaptor family member 1 220122_at 0 MCTP1 NM_024717; multiple C2 domains, transmembrane 1 NM_001002796 220308_at 0 CCDC19 NM_012337 coiled-coil domain containing 19 220319_s_at 1 MYLIP NM_013262 myosin regulatory light chain interacting protein 220646_s_at 0 KLRF1 NM_016523 killer cell lectin-like receptor subfamily F, member 1 220765_s_at 0 LIMS2 NM_017980; LIM and senescent cell antigen-like domains 2 NM_001161404; NM_001161403; NM_001136037 220935_s_at 0 CDK5RAP2 NM_018249; CDK5 regulatory subunit associated protein 2 NM_001011649 221032_s_at 0 TMPRSS5 NM_030770 transmembrane protease, serine 5 221142_s_at 0 PECR NM_018441 peroxisomal trans-2-enoyl-CoA reductase 221211_s_at 0 C21orf7 NM_020152 chromosome 21 open reading frame 7 221491_x_at 0 HLA-DRB1 XM_002346768; major histocompatibility complex, class II, DR beta 3 /// HLA- NM_022555; DRB3 /// XM_002346769 HLA-DRB4 221874_at 0 KIAA1324 NM_020775 KIAA1324 221964_at 0 TULP3 NM_001160408; tubby like protein 3 NM_003324 222059_at 0 ZNF335 NM_022095 zinc finger protein 335 222186_at 1 ZA20D3 NM_019006 zinc finger, AN1-type domain 6 222297_x_at 0 RPL18 ribosomal protein L18 222330_at 0 PDE3B NM_000922 phosphodiesterase 3B, cGMP-inhibited 320_at 0 PEX6 NM_000287 peroxisomal biogenesis factor 6 44673_at 0 SN NM_023068 sialic acid binding Ig-like lectin 1, sialoadhesin 49329_at 1 KLHL22 NM_032775 kelch-like 22 (Drosophila) 49452_at 1 ACACB NM_001093 acetyl-Coenzyme A carboxylase beta 215185_at 0 LOC441468 AFFX- 0 GAPDH HUMGAPDH/ M33197_M_at 206512_at 0 U2AF1L1 ambiguous (pending) 211781_x_at 0 216635_at 0 216943_at 0 217079_at 0 220352_x_at 0 Methods of Treating a Subject with an ARI

Another aspect of the present disclosure provides a method of treating an acute respiratory infection (ARI) whose etiology is unknown in a subject, said method comprising, consisting of, or consisting essentially of (a) obtaining a biological sample from the subject; (b) determining the gene expression profile of the subject from the biological sample by evaluating the expression levels of pre-defined sets of genes (e.g., one, two or three or more signatures); (c) normalizing gene expression levels as required for the technology used to make said measurement to generate a normalized value; (d) entering the normalized value into a bacterial classifier, a viral classifier and non-infectious illness classifier (i.e., predictors) that have pre-defined weighting values (coefficients) for each of the genes in each signature; (e) comparing the output of the classifiers to pre-defined thresholds, cut-off values, or ranges of values that indicate likelihood of infection; (f) classifying the sample as being of bacterial etiology, viral etiology, or noninfectious illness; and (g) administering to the subject an appropriate treatment regimen as identified by step (f).

In some embodiments, step (g) comprises administering an antibacterial therapy when the etiology of the ARI is determined to be bacterial. In other embodiments, step (g) comprises administering an antiviral therapy when the etiology of the ARI is determined to be viral.

After the etiology of the ARI of the subject has been determined, she may undergo treatment, for example anti-viral therapy if the ARI is determined to be viral, and/or she may be quarantined to her home for the course of the infection. Alternatively, bacterial therapy regimens may be administered (e.g., administration of antibiotics) if the ARI is determined to be bacterial. Those subjects classified as non-infectious illness may be sent home or seen for further diagnosis and treatment (e.g., allergy, asthma, etc.).

The person performing the peripheral blood sample need not perform the comparison, however, as it is contemplated that a laboratory may communicate the gene expression levels of the classifiers to a medical practitioner for the purpose of identifying the etiology of the ARI and for the administration of appropriate treatment. Additionally, it is contemplated that a medical professional, after examining a patient, would order an agent to obtain a peripheral blood sample, have the sample assayed for the classifiers, and have the agent report patient's etiological status to the medical professional. Once the medical professional has obtained the etiology of the ARI, the medical professional could order suitable treatment and/or quarantine.

The methods provided herein can be effectively used to diagnose the etiology of illness in order to correctly treat the patient and reduce inappropriate use of antibiotics. Further, the methods provided herein have a variety of other uses, including but not limited to, (1) a host-based test to detect individuals who have been exposed to a pathogen and have impending, but not symptomatic, illness (e.g., in scenarios of natural spread of diseases through a population but also in the case of bioterrorism); (2) a host-based test for monitoring response to a vaccine or a drug, either in a clinical trial setting or for population monitoring of immunity; (3) a host-based test for screening for impending illness prior to deployment (e.g., a military deployment or on a civilian scenario such as embarkation on a cruise ship); and (4) a host-based test for the screening of livestock for ARIs (e.g., avian flu and other potentially pandemic viruses).

Another aspect of the present disclosure provides a kit for determining the etiology of an acute respiratory infection (ARI) in a subject comprising, consisting of, or consisting essentially of (a) a means for extracting a biological sample; (b) a means for generating one or more arrays consisting of a plurality of synthetic oligonucleotides with regions homologous to a group of gene transcripts as taught herein; and (c) instructions for use.

Yet another aspect of the present disclosure provides a method of using a kit for assessing the acute respiratory infection (ARI) classifier comprising, consisting of, or consisting essentially of: (a) generating one or more arrays consisting of a plurality of synthetic oligonucleotides with regions homologous to a a group of gene transcripts as taught herein; (b) adding to said array oligonucleotides with regions homologous to normalizing genes; (c) obtaining a biological sample from a subject suffering from an acute respiratory infection (ARI); (d) isolating RNA from said sample to create a transcriptome; (e) measuring said transcriptome on said array; (f) normalizing the measurements of said transcriptome to the normalizing genes, electronically transferring normalized measurements to a computer to implement the classifier algorithm(s), (g) generating a report; and optionally (h) administering an appropriate treatment based on the results.

Classification Systems

With reference to FIG. 11, a classification system and/or computer program product 1100 may be used in or by a platform, according to various embodiments described herein. A classification system and/or computer program product 1100 may be embodied as one or more enterprise, application, personal, pervasive and/or embedded computer systems that are operable to receive, transmit, process and store data using any suitable combination of software, firmware and/or hardware and that may be standalone and/or interconnected by any conventional, public and/or private, real and/or virtual, wired and/or wireless network including all or a portion of the global communication network known as the Internet, and may include various types of tangible, non-transitory computer readable medium.

As shown in FIG. 11, the classification system 1100 may include a processor subsystem 1140, including one or more Central Processing Units (CPU) on which one or more operating systems and/or one or more applications run. While one processor 1140 is shown, it will be understood that multiple processors 1140 may be present, which may be either electrically interconnected or separate. Processor(s) 1140 are configured to execute computer program code from memory devices, such as memory 1150, to perform at least some of the operations and methods described herein, and may be any conventional or special purpose processor, including, but not limited to, digital signal processor (DSP), field programmable gate array (FPGA), application specific integrated circuit (ASIC), and multi-core processors.

The memory subsystem 1150 may include a hierarchy of memory devices such as Random Access Memory (RAM), Read-Only Memory (ROM), Erasable Programmable Read-Only Memory (EPROM) or flash memory, and/or any other solid state memory devices.

A storage circuit 1170 may also be provided, which may include, for example, a portable computer diskette, a hard disk, a portable Compact Disk Read-Only Memory (CDROM), an optical storage device, a magnetic storage device and/or any other kind of disk- or tape-based storage subsystem. The storage circuit 1170 may provide non-volatile storage of data/parameters/classifiers for the classification system 1100. The storage circuit 1170 may include disk drive and/or network store components. The storage circuit 1170 may be used to store code to be executed and/or data to be accessed by the processor 1140. In some embodiments, the storage circuit 1170 may store databases which provide access to the data/parameters/classifiers used for the classification system 1110 such as the signatures, weights, thresholds, etc. Any combination of one or more computer readable media may be utilized by the storage circuit 1170. The computer readable media may be a computer readable signal medium or a computer readable storage medium. A computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage medium would include the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. As used herein, a computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device.

An input/output circuit 1160 may include displays and/or user input devices, such as keyboards, touch screens and/or pointing devices. Devices attached to the input/output circuit 1160 may be used to provide information to the processor 1140 by a user of the classification system 1100. Devices attached to the input/output circuit 1160 may include networking or communication controllers, input devices (keyboard, a mouse, touch screen, etc.) and output devices (printer or display). The input/output circuit 1160 may also provide an interface to devices, such as a display and/or printer, to which results of the operations of the classification system 1100 can be communicated so as to be provided to the user of the classification system 1100.

An optional update circuit 1180 may be included as an interface for providing updates to the classification system 1100. Updates may include updates to the code executed by the processor 1140 that are stored in the memory 1150 and/or the storage circuit 1170. Updates provided via the update circuit 1180 may also include updates to portions of the storage circuit 1170 related to a database and/or other data storage format which maintains information for the classification system 1100, such as the signatures, weights, thresholds, etc.

The sample input circuit 1110 of the classification system 1100 may provide an interface for the platform as described hereinabove to receive biological samples to be analyzed. The sample input circuit 1110 may include mechanical elements, as well as electrical elements, which receive a biological sample provided by a user to the classification system 1100 and transport the biological sample within the classification system 1100 and/or platform to be processed. The sample input circuit 1110 may include a bar code reader that identifies a bar-coded container for identification of the sample and/or test order form. The sample processing circuit 1120 may further process the biological sample within the classification system 1100 and/or platform so as to prepare the biological sample for automated analysis. The sample analysis circuit 1130 may automatically analyze the processed biological sample. The sample analysis circuit 1130 may be used in measuring, e.g., gene expression levels of a pre-defined set of genes with the biological sample provided to the classification system 1100. The sample analysis circuit 1130 may also generate normalized gene expression values by normalizing the gene expression levels. The sample analysis circuit 1130 may retrieve from the storage circuit 1170 a bacterial acute respiratory infection (ARI) classifier, a viral ARI classifier and a non-infectious illness classifier, these classifier(s) comprising pre-defined weighting values (i.e., coefficients) for each of the genes of the pre-defined set of genes. The sample analysis circuit 1130 may enter the normalized gene expression values into one or more acute respiratory illness classifiers selected from the bacterial acute respiratory infection (ARI) classifier, the viral ARI classifier and the non-infectious illness classifier. The sample analysis circuit 1130 may calculate an etiology probability for one or more of a bacterial ARI, viral ARI and non-infectious illness based upon said classifier(s) and control output, via the input/output circuit 1160, of a determination whether the acute respiratory illness in the subject is bacterial in origin, viral in origin, non-infectious in origin, or some combination thereof.

The sample input circuit 1110, the sample processing circuit 1120, the sample analysis circuit 1130, the input/output circuit 1160, the storage circuit 1170, and/or the update circuit 1180 may execute at least partially under the control of the one or more processors 1140 of the classification system 1100. As used herein, executing “under the control” of the processor 1140 means that the operations performed by the sample input circuit 1110, the sample processing circuit 1120, the sample analysis circuit 1130, the input/output circuit 1160, the storage circuit 1170, and/or the update circuit 1180 may be at least partially executed and/or directed by the processor 1140, but does not preclude at least a portion of the operations of those components being separately electrically or mechanically automated. The processor 1140 may control the operations of the classification system 1100, as described herein, via the execution of computer program code.

Computer program code for carrying out operations for aspects of the present disclosure may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, Scala, Smalltalk, Eiffel, JADE, Emerald, C++, C#, VB.NET, Python or the like, conventional procedural programming languages, such as the “C” programming language, Visual Basic, Fortran 2003, Perl, COBOL 2002, PHP, ABAP, dynamic programming languages such as Python, Ruby and Groovy, or other programming languages. The program code may execute entirely on the classification system 1100, partly on the classification system 1100, as a stand-alone software package, partly on the classification system 1100 and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the classification system 1100 through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider) or in a cloud computer environment or offered as a service such as a Software as a Service (SaaS).

In some embodiments, the system includes computer readable code that can transform quantitative, or semi-quantitative, detection of gene expression to a cumulative score or probability of the etiology of the ARI.

In some embodiments, the system is a sample-to-result system, with the components integrated such that a user can simply insert a biological sample to be tested, and some time later (preferably a short amount of time, e.g., 30 or 45 minutes, or 1, 2, or 3 hours, up to 8, 12, 24 or 48 hours) receive a result output from the system.

It is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

It also is understood that any numerical range recited herein includes all values from the lower value to the upper value. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The following examples are illustrative only and are not intended to be limiting in scope.

EXAMPLES Example 1 Host Gene Expression Classifiers Diagnose Acute Respiratory Illness Etiology

Acute respiratory infections due to bacterial or viral pathogens are among the most common reasons for seeking medical care. Current pathogen-based diagnostic approaches are not reliable or timely, thus most patients receive inappropriate antibiotics. Host response biomarkers offer an alternative diagnostic approach to direct antimicrobial use.

We asked whether host gene expression patterns discriminate infectious from non-infectious causes of illness in the acute care setting. Among those with acute respiratory infection, we determined whether infectious illness is due to viral or bacterial pathogens.

The samples that formed the basis for discovery were drawn from an observational, cohort study conducted at four tertiary care hospital emergency departments and a student health facility. 44 healthy controls and 273 patients with community-onset acute respiratory infection or non-infectious illness were selected from a larger cohort of patients with suspected sepsis (CAPSOD study). Mean age was 45 years and 45% of participants were male. Further demographic information may be found in Table 1 of Tsalik et al. (2016) Sci Transl Med 9(322):1-9, which is incorporated by reference herein.

Clinical phenotypes were adjudicated through manual chart review. Routine microbiological testing and multiplex PCR for respiratory viral pathogens were performed. Peripheral whole blood gene expression was measured using microarrays. Sparse logistic regression was used to develop classifiers of bacterial vs. viral vs. non-infectious illness. Five independently derived datasets including 328 individuals were used for validation.

Gene expression-based classifiers were developed for bacterial acute respiratory infection (71 probes), viral acute respiratory infection (33 probes), or a non-infectious cause of illness (26 probes). The three classifiers were applied to 273 patients where class assignment was determined by the highest predicted probability. Overall accuracy was 87% (23 8/273 concordant with clinical adjudication), which was more accurate than procalcitonin (78%, p<0.03) and three published classifiers of bacterial vs. viral infection (78-83%). The classifiers developed here externally validated in five publicly available datasets (AUC 0.90-0.99). We compared the classification accuracy of the host gene expression-based tests to procalcitonin and clinically adjudicated diagnoses, which included bacterial or viral acute respiratory infection or non-infectious illness.

The host's peripheral blood gene expression response to infection offers a diagnostic strategy complementary to those already in use.⁸ This strategy has successfully characterized the host response to viral⁸⁻¹³ and bacterial ARI^(11,14). Despite these advances, several issues preclude their use as diagnostics in patient care settings. An important consideration in the development of host-based molecular signatures is that they be developed in the intended use population.¹⁵ However, nearly all published gene expression-based ARI classifiers used healthy individuals as controls and focused on small or homogeneous populations and are thus not optimized for use in acute care settings where patients present with undifferentiated symptoms. Furthermore, the statistical methods used to identify gene-expression classifiers often include redundant genes based on clustering, univariate testing, or pathway association. These strategies identify relevant biology but do not maximize diagnostic performance. An alternative, as exemplified here, is to combine genes from unrelated pathways to generate a more informative classifier.

Methods Classifier Derivation Cohorts

Studies were approved by relevant Institutional Review Boards, and in accord with the Declaration of Helsinki. All subjects or their legally authorized representatives provided written informed consent.

Patients with community-onset, suspected infection were enrolled in the Emergency Departments of Duke University Medical Center (DUMC; Durham, N.C.), the Durham VA Medical Center (DVAMC; Durham, N.C.), or Henry Ford Hospital (Detroit, Mich.) as part of the Community Acquired Pneumonia & Sepsis Outcome Diagnostics study (Clinical Trials Identifier No. NCT00258869).¹⁶⁻¹⁹ Additional patients were enrolled through UNC Health Care Emergency Department (UNC; Chapel Hill, N.C.) as part of the Community Acquired Pneumonia and Sepsis Study. Patients were eligible if they had a known or suspected infection and if they exhibited two or more Systemic Inflammatory Response Syndrome (SIRS) criteria.²⁰ ARI cases included patients with upper or lower respiratory tract symptoms, as adjudicated by emergency medicine (SWG, EBQ) or infectious diseases (ELT) physicians. Adjudications were based on retrospective, manual chart reviews performed at least 28 days after enrollment and prior to any gene expression-based categorization, using previously published criteria.¹⁷ The totality of information used to support these adjudications would not have been available to clinicians at the time of their evaluation. Seventy patients with microbiologically confirmed bacterial ARI were identified including four with pharyngitis and 66 with pneumonia. Microbiological etiologies were determined using conventional culture of blood or respiratory samples, urinary antigen testing (Streptococcus or Legionella), or with serological testing (Mycoplasma) Patients with viral ARI (n=115) were ascertained based on identification of a viral etiology and compatible symptoms. In addition, 48 students at Duke University as part of the DARPA Predicting Health and Disease study with definitive viral ARI using the same adjudication methods were included. The ResPlex II v2.0 viral PCR multiplex assay (Qiagen; Hilden, Germany) augmented clinical testing for viral etiology identification. This panel detects influenza A and B, adenovirus (B, E), parainfluenza 1-4, respiratory syncytial virus A and B, human metapneumovirus, human rhinovirus, coronavirus (229E, OC43, NL63, HKU1), coxsackie/echo virus, and bocavirus. Upon adjudication, a subset of enrolled patients were determined to have non-infectious illness (n=88) (Table 8). The determination of “non-infectious illness” was made only when an alternative diagnosis was established and results of any routinely ordered microbiological testing failed to support an infectious etiology. Lastly, healthy controls (n=44; median age 30 years; range 23-59) were enrolled as part of a study on the effect of aspirin on platelet function among healthy volunteers without symptoms, where gene expression analyses was performed on pre-aspirin challenge time points.²¹

Procalcitonin Measurement

Concentrations were measured at different stages during the study and as a result, different platforms were utilized based on availability. Some serum measurements were made on a Roche Elecsys 2010 analyzer (Roche Diagnostics, Laval, Canada) by electrochemiluminescent immunoassay. Additional serum measurements were made using the miniVIDAS immunoassay (bioMerieux, Durham N.C., USA). When serum was unavailable, measurements were made by the Phadia Immunology Reference Laboratory in plasma-EDTA by immunofluorescence using the B⋅R⋅A⋅H⋅M⋅S PCT sensitive KRYPTOR (Thermo Fisher Scientific, Portage Mich., USA). Replicates were performed for some paired serum and plasma samples, revealing equivalence in concentrations. Therefore, all procalcitonin measurements were treated equivalently, regardless of testing platform.

Microarray Generation

At initial clinical presentation, patients were enrolled and samples collected for analysis. After adjudications were performed as described above, 317 subjects with clear clinical phenotypes were selected for gene expression analysis. Total RNA was extracted from human blood using the PAXgene Blood RNA Kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocol. RNA quantity and quality were assessed using the Nanodrop spectrophotometer (Thermo Scientific, Waltham, Mass.) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, Calif.), respectively. Microarrays were RMA-normalized. Hybridization and data collection were performed at Expression Analysis (Durham, N.C.) using the GeneChip Human Genome U133A 2.0 Array (Affymetrix, Santa Clara, Calif.) according to the Affymetrix Technical Manual.

Statistical Analysis

The transcriptomes of 317 subjects (273 ill patients and 44 healthy volunteers) were measured in two microarray batches with seven overlapping samples (GSE63990). Exploratory principal component analysis and hierarchical clustering revealed substantial batch differences. These were corrected by first estimating and removing probe-wise mean batch effects using the Bayesian fixed effects model. Next, we fitted a robust linear regression model with Huber loss function using seven overlapping samples, which was used to adjust the remaining expression values.

Sparse classification methods such as sparse logistic regression perform classification and variable selection simultaneously while reducing over-fitting risk.²¹ Therefore, separate gene selection strategies such as univariate testing or sparse factor models are unnecessary. Here, a sparse logistic regression model was fitted independently to each of the binary tasks using the 40% of probes with the largest variance after batch correction.²² Specifically, we used a Lasso regularized generalized linear model with binomial likelihood with nested cross-validation to select for the regularization parameters. Code was written in Matlab using the Glmnet toolbox. This generated Bacterial ARI, Viral ARI, and Non-Infectious Illness classifiers. Provided that each binary classifier estimates class membership probabilities (e.g., probability of bacterial vs. either viral or non-infectious in the case of the Bacterial ARI classifier), we can combine the three classifiers into a single decision model (termed the ARI classifier) by following a one-versus-all scheme whereby largest membership probability assigns class label.²¹ Classification performance metrics included area-under-the-receiving-operating-characteristic-curve (AUC) for binary outcomes and confusion matrices for ternary outcomes.²³

Validation

The ARI classifier was validated using leave-one-out cross-validation in the same population from which it was derived. Independent, external validation occurred using publically available human gene expression datasets from 328 individuals (GSE6269, GSE42026, GSE40396, GSE20346, and GSE42834). Datasets were chosen if they included at least two clinical groups (bacterial ARI, viral ARI, or non-infectious illness). To match probes across different microarray platforms, each ARI classifier probe was converted to gene symbols, which were used to identify corresponding target microarray probes.

Results Bacterial ARI, Viral ARI, and Non-Infectious Illness Classifiers

In generating host gene expression-based classifiers that distinguish between clinical states, all relevant clinical phenotypes should be represented during the model training process. This imparts specificity, allowing the model to be applied to these included clinical groups but not to clinical phenotypes that were absent from model training.¹⁵ The target population for an ARI diagnostic not only includes patients with viral and bacterial etiologies, but must also distinguish from the alternative—those without bacterial or viral ARI. Historically, healthy individuals have served as the uninfected control group. However, this fails to consider how patients with non-infectious illness, which can present with similar clinical symptoms, would be classified, serving as a potential source of diagnostic error. To our knowledge, no ARI gene-expression based classifier has included ill, uninfected controls in its derivation. We therefore enrolled a large, heterogeneous population of patients at initial clinical presentation with community-onset viral ARI (n=115), bacterial ARI (n=70), or non-infectious illness (n=88) (Table 8). We also included a healthy adult control cohort (n=44) to define the most appropriate control population for ARI classifier development.

We first determined whether a gene expression classifier derived with healthy individuals as controls could accurately classify patients with non-infectious illness. Array data from patients with bacterial ARI, viral ARI, and healthy controls were used to generate gene expression classifiers for these conditions. Leave-one-out cross validation revealed highly accurate discrimination between bacterial ARI (AUC 0.96), viral ARI (AUC 0.95), and healthy (AUC 1.0) subjects for a combined accuracy of 90% (FIG. 7). However, when the classifier was applied to ill-uninfected patients, 48/88 were identified as bacterial, 35/88 as viral, and 5/88 as healthy. This highlighted that healthy individuals are a poor substitute for patients with non-infectious illness in the biomarker discovery process.

Consequently, we re-derived an ARI classifier using a non-infectious illness control rather than healthy. Specifically, array data from these three groups was used to generate three gene-expression classifiers of host response to bacterial ARI, viral ARI, and non-infectious illness (FIG. 5). Specifically, the Bacterial ARI classifier was tasked with positively identifying those with bacterial ARI vs. either viral ARI or non-infectious illnesses. The Viral ARI classifier was tasked with positively identifying those with viral ARI vs. bacterial ARI or non-infectious illnesses. The Non-Infectious Illness classifier was not generated with the intention of positively identifying all non-infectious illnesses, which would require an adequate representation of all such cases.

Rather, it was generated as an alternative category, so that patients without bacterial or viral ARI could be assigned accordingly. Moreover, we hypothesized that such ill but non-infected patients were more clinically relevant controls because healthy people are unlikely to be the target for such a classification task.

Six statistical strategies were employed to generate these gene-expression classifiers: linear support vector machines, supervised factor models, sparse multinomial logistic regression, elastic nets, K-nearest neighbor, and random forests. All performed similarly although sparse logistic regression required the fewest number of classifier genes and outperformed other strategies by a small margin (data not shown). We also compared a strategy that generated three separate binary classifiers to a single multinomial classifier that would simultaneously assign a given subject to one of the three clinical categories. This latter approach required more genes and achieved an inferior accuracy. Consequently, we applied a sparse logistic regression model to define Bacterial ARI, Viral ARI, and Non-Infectious Illness classifiers containing 71, 33 and 26 probe signatures, respectively. Probe and classifier weights are shown in Table 9.

Clinical decision making is infrequently binary, requiring the simultaneous distinction of multiple diagnostic possibilities. We applied all three classifiers, collectively defined as the ARI classifier, using leave-one-out cross-validation to assign probabilities of bacterial ARI, viral ARI, and non-infectious illness (FIG. 6). These conditions are not mutually exclusive. For example, the presence of a bacterial ARI does not preclude a concurrent viral ARI or non-infectious disease. Moreover, the assigned probability represents the extent to which the patient's gene expression response matches that condition's canonical signature. Since each signature intentionally functions independently of the others, the probabilities are not expected to sum to one. To simplify classification, the highest predicted probability determined class assignment. Overall classification accuracy was 87% (238/273 were concordant with adjudicated phenotype).

Bacterial ARI was identified in 58/70 (83%) patients and excluded 179/191 (94%) without bacterial infection. Viral ARI was identified in 90% (104/115) and excluded in 92% (145/158) of cases. Using the non-infectious illness classifier, infection was excluded in 86% of cases (76/88). Sensitivity analyses was performed for positive and negative predictive values for all three classifiers given that prevalence can vary for numerous reasons including infection type, patient characteristics, or location (FIG. 8). For both bacterial and viral classification, predictive values remained high across a range of prevalence estimates, including those typically found for ARI.

To determine if there was any effect of age, we included it as a variable in the classification scheme. This resulted in two additional correct classifications, likely due to the over-representation of young people in the viral ARI cohort. However, we observed no statistically significant differences between correctly and incorrectly classified subjects due to age (Wilcoxon rank sum p=0.17).

We compared this performance to procalcitonin, a widely used biomarker specific for bacterial infection. Procalcitonin concentrations were determined for the 238 subjects where samples were available and compared to ARI classifier performance for this subgroup. Procalcitonin concentrations >0.25 μg/L assigned patients as having bacterial ARI, whereas values ≤0.25 μg/L assigned patients as non-bacterial, which could be either viral ARI or non-infectious illness. Procalcitonin correctly classified 186 of 238 patients (78%) compared to 204/238 (86%) using the ARI classifier (p=0.03). However, accuracy for the two strategies varied depending on the classification task. For example, performance was similar in discriminating viral from bacterial ARI. Procalcitonin correctly classified 136/155 (AUC 0.89) compared to 140/155 for the ARI classifier (p-value=0.65 using McNemar's test with Yates correction). However, the ARI classifier was significantly better than procalcitonin in discriminating bacterial ARI from non-infectious illness [105/124 vs. 79/124 (AUC 0.72); p-value<0.001], and discriminating bacterial ARI from all other etiologies including viral and non-infectious etiologies [215/238 vs. 186/238 (AUC 0.82); p-value=0.02].

We next compared the ARI classifier to three published gene expression classifiers of bacterial vs. viral infection, each of which was derived without uninfected ill controls. These included a 35-probe classifier (Ramilo) derived from children with influenza or bacterial sepsis¹¹; a 33-probe classifier (Hu) derived from children with febrile viral illness or bacterial infection¹⁴; and a 29-probe classifier (Parnell) derived from adult ICU patients with community-acquired pneumonia or influenza¹². We hypothesized that classifiers generated using only patients with viral or bacterial infection would perform poorly when applied to a clinically relevant population that included ill but uninfected patients. Specifically, when presented with an individual with neither a bacterial nor a viral infection, the previously published classifiers would be unable to accurately assign that individual to a third, alternative category. We therefore applied the derived as well as published classifiers to our 273-patient cohort. Discrimination between bacterial ARI, viral ARI, and non-infectious illness was better with the derived ARI classifier (McNemar's test with Yates correction, p=0.002 vs. Ramilo; p=0.0001 vs. Parnell; and p=0.08 vs. Hu) (Table 6).^(24,25) This underscores the importance of deriving gene-expression classifiers in a cohort representative of the intended use population, which in the case of ARI should include non-infectious illness.¹⁵

Discordant Classifications

To better understand ARI classifier performance, we individually reviewed the 35 discordant cases. Nine adjudicated bacterial infections were classified as viral and three as non-infectious illness. Four viral infections were classified as bacterial and seven as non-infectious. Eight non-infectious cases were classified as bacterial and four as viral. We did not observe a consistent pattern among discordant cases, however, notable examples included atypical bacterial infections. One patient with M pneumoniae based on serological conversion and one of three patients with Legionella pneumonia were classified as viral ARI. Of six patients with non-infectious illness due to autoimmune or inflammatory diseases, only one adjudicated to have Still's disease was classified as having bacterial infection. See also eTable 3 of Tsalik et al. (2016) Sci Transl Med 9(322):1-9, which is incorporated by reference herein.

External Validation

Generating classifiers from high dimensional, gene expression data can result in over-fitting. We therefore validated the ARI classifier in silico using gene expression data from 328 individuals, represented in five available datasets (GSE6269, GSE42026, GSE40396, GSE20346, and GSE42834). These were chosen because they included at least two relevant clinical groups, varying in age, geographic distribution, and illness severity (Table 7). Applying the ARI classifier to four datasets with bacterial and viral ARI, AUC ranged from 0.90-0.99. Lastly, GSE42834 included patients with bacterial pneumonia (n=19), lung cancer (n=16), and sarcoidosis (n=68). Overall classification accuracy was 96% (99/103) corresponding to an AUC of 0.99. GSE42834 included five subjects with bacterial pneumonia pre- and post-treatment. All five demonstrated a treatment-dependent resolution of the bacterial infection. See also eFigures 3-8 of Tsalik et al. (2016) Sci Transl Med 9(322):1-9, which is incorporated by reference herein.

Biological Pathways

The sparse logistic regression model that generated the classifiers penalizes selection of genes from a given pathway if there is no additive diagnostic value. Consequently, conventional gene enrichment pathway analysis is not appropriate to perform. Moreover, such conventional gene enrichment analyses have been described.^(9,12,14,28,29) Instead a literature review was performed for all classifier genes (Table 10). Overlap between Bacterial, Viral, and Non-infectious Illness Classifiers is shown in FIG. 9.

The Viral classifier included known anti-viral response categories such as interferon response, T-cell signaling, and RNA processing. The Viral classifier had the greatest representation of RNA processing pathways such as KPNB1, which is involved in nuclear transport and is co-opted by viruses for transport of viral proteins and genomes.^(26,27) Its downregulation suggests it may play an antiviral role in the host response.

The Bacterial classifier encompassed the greatest breadth of cellular processes, notably cell cycle regulation, cell growth, and differentiation. The Bacterial classifier included genes important in T-, B-, and NK-cell signaling. Unique to the Bacterial classifier were genes involved in oxidative stress, and fatty acid and amino acid metabolism, consistent with sepsis-related metabolic perturbations.²⁸

Summary of Clinical Applicability

We determined that host gene expression changes are exquisitely specific to the offending pathogen class and can be used to discriminate common etiologies of respiratory illness. This creates an opportunity to develop and utilize gene expression classifiers as novel diagnostic platforms to combat inappropriate antibiotic use and emerging antibiotic resistance. Using sparse logistic regression, we developed host gene expression profiles that accurately distinguished between bacterial and viral etiologies in patients with acute respiratory symptoms (external validation AUC 0.90-0.99). Deriving the ARI classifier with a non-infectious illness control group imparted a high negative predictive value across a wide range of prevalence estimates.

Respiratory tract infections caused 3.2 million deaths worldwide and 164 million disability-adjusted life years lost in 2011, more than any other cause.^(1,2) Despite a viral etiology in the majority of cases, 73% of ambulatory care patients in the U.S. with acute respiratory infection (ARI) are prescribed an antibiotic, accounting for 41% of all antibiotics prescribed in this setting.^(3,4) Even when a viral pathogen is microbiologically confirmed, this does not exclude a possible concurrent bacterial infection leading to antimicrobial prescribing “just in case”. This empiricism drives antimicrobial resistance^(5,6), recognized as a national security priority.⁷ The encouraging metrics provided in this example provide an opportunity to provide clinically actionable results which will optimize treatment and mitigate emerging antibiotic resistance.

Several studies made notable inroads in developing host-response diagnostics for ARI. This includes response to respiratory viruses^(8,10-12,14), bacterial etiologies in an ICU population^(12,30), and tuberculosis³¹⁻³³. Typically, these define host response profiles compared to the healthy state, offering valuable insights into host biology.^(16,34,35) However, these gene lists are suboptimal with respect to a diagnostic application because the gene expression profiles that are a component of the diagnostic is not representative of the population for which the test will be applied.¹⁵ Healthy individuals do not present with acute respiratory complaints, thus they are excluded from the host-response diagnostic development reported herein.

Including patients with bacterial and viral infections allows for the distinction between these two states but does not address how to classify non-infectious illness. This phenotype is important to include because patients present with infectious and non-infectious etiologies that may share symptoms. That is, symptoms may not provide a clinician with a high degree of diagnostic certainty. The current approach, which uniquely appreciates the necessity of including the three most likely states for ARI symptoms, can be applied to an undifferentiated clinical population where such a test is in greatest need.

The small number of discordant classifications occurred may have arisen either from errors in classification or clinical phenotyping. Errors in clinical phenotyping can arise from a failure to identify causative pathogens due to limitations in current microbiological diagnostics. Alternatively, some non-infectious disease processes may in fact be infection-related through mechanisms that have yet to be discovered. Discordant cases were not clearly explained by a unifying variable such as pathogen type, syndrome, or patient characteristic. As such, the gene expression classifiers presented herein may be impacted by other factors including patient-specific variables (e.g., treatment, comorbidity, duration of illness); test-specific variables (e.g., sample processing, assay conditions, RNA quality and yield); or as-of-yet unidentified variables.

Example 2 Classification Performance in Patients with Co-Infection Defined by the Identification of Bacterial and Viral Pathogens

In addition to determining that age did not significantly impact classification accuracy, we assessed whether severity of illness or etiology of SIRS affected classification. Patients with viral ARI tended to be less ill, as evidenced by lower rate of hospitalization. In the various cohorts, hospitalization was used as a marker of disease severity and its impact on classification performance was assessed. This test revealed no difference (Fisher's exact test p-value of 1). In addition, the SIRS control cohort included subjects with both respiratory and non-respiratory etiologies. We assessed whether classification was different in subjects with respiratory vs. non-respiratory SIRS and determined it was not (Fisher's exact test p-value of 0.1305).

Some patients with ARI will have both bacterial and viral pathogens identified, often termed co-infection. However, it is unclear how the host responds in such situations. Illness may be driven by the bacteria, the virus, both, or neither at different times in the patient's clinical course. We therefore determined how the bacterial and viral ARI classifiers performed in a population with bacterial and viral co-identification. GSE60244 included bacterial pneumonia (n=22), viral respiratory tract infection (n=71), and bacterial/viral co-identification (n=25). The co-identification group was defined by the presence of both bacterial and viral pathogens without further subcategorization as to the likelihood of bacterial or viral disease. We trained classifiers on subjects in GSE60244 with bacterial or viral infection and then validated in those with co-identification (FIG. 10). A host response was considered positive above a probability threshold of 0.5. We observed all four possible categories. Six of 25 subjects had a positive bacterial signature; 14/25 had a viral response; 3/25 had positive bacterial and viral signatures; and 2/25 had neither.

The major clinical decision faced by clinicians is whether or not to prescribe antibacterials. A simpler diagnostic strategy might focus only on the probability of bacterial ARI according to the result from the Bacterial ARI classifier. However, there is value in providing information about viral or non-infectious alternatives. For example, the confidence to withhold antibacterials in a patient with a low probability of bacterial ARI can be enhanced by a high probability of an alternative diagnosis. Further, a full diagnostic report could identify concurrent illness that a single classifier would miss. We observed this when validating in a population with bacterial and viral co-identification. These patients are more commonly referred to as “co-infected.” To have infection, there must be a pathogen, a host, and a maladaptive interaction between the two. Simply identifying bacterial and viral pathogens should not imply co-infection. Although we cannot know the true infection status in the 25 subjects tested, who had evidence of bacterial/viral co-identification, the host response classifiers suggest the existence of multiple host-response states. FIG. 10 is an informative representation of infection status, which could be used by a clinician to diagnose the etiology of ARI.

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TABLE 8 Etiological causes of illness for subjects with viral ARI, bacterial ARI, and non-nfectious illness. Number of subjects Total Cohort 273 All Viral ARI 115 Coronavirus 7 Coxsackievirus/Echovirus 3 Cytomegalovirus 1 Enterovirus 20 Human Metapneumovirus 9 Influenza, non-typed 7 Influenza A, non-subtyped 6 Influenza A, 2009 H1N1 37 Parainfluenza 1 Polymicrobial (Coronavirus, Rhinovirus, Coxsackievirus/ 1 Echovirus) Rhinovirus 19 Respiratory Syncitial Virus 6 All Bacterial ARI 70 Bacillus species^(a) 1 Bordetella bronchiseptica 1 Enterobacter aerogenes 1 Escherichia coli 1 Haemophilus influenza 3 Legionella sp. 3 Mycoplasma pneumoniae 1 Pasteurella multocida 1 Polymicrobial 11 Pantoea sp.; Coagulase negative Staphylococcus 1 Pseudomonas aeruginosa; Alcaligenes xylosoxidans 1 Pseudomonas aeruginosa; Serratia marcescens 1 Staphylococcus aureus; Haemophilus influenzae 2 Staphylococcus aureus; Proteus mirabilis 1 Staphylococcus aureus; Viridans Group Streptococcus; 1 Escherichia coli Streptococcus pneumoniae; Haemophilus sp. 1 Streptococcus pneumoniae; Staphylococcus aureus 3 Proteus mirabilis 1 Pseudomonas aeruginosa 4 Staphylococcus aureus 7 Streptococcus pneumoniae 30 Streptococcus pyogenes 4 Viridans Group Streptococcus 1 All Non-Infectious Illness 88 Acute Renal Failure; Hypovolemia 1 Alcohol intoxication; Spinal cord stenosis; Hyperglycemia 1 Arrhythmia 2 Asthma 1 AV Graft Pseudoaneurysm and Thrombus 1 Brain Metastases with Vasogenic Edema 1 Cerebrovascular Accident 1 Chest Pain 2 Cocaine Intoxication 1 Congestive Heart Failure 13 Congestive Heart Failure; Amiodarone Toxicity 1 Congestive Heart Failure; Arrhythmia 1 Chronic Obstructive Pulmonary Disease 5 Cryptogenic Organizing Pneumonia 1 Emphysema 1 Gastrointestinal Hemorrhage 3 Hematoma in Leg 1 Hemochromatosis; Abdominal Pain and Peritoneal Dialysis 1 Hemothorax 1 Heroin Overdose 1 Hyperglycemia 2 Hypertensive Emergency 3 Hypertensive Emergency with Pulmonary Edema 1 Hypovolemia 2 Infarcted Uterine Fibroid 1 Lung Cancer; Coronary Artery Disease 1 Lung Cancer; Hemoptysis 1 Mitochondrial Disorder; Acidosis 1 Myocardial Infarction 2 Myocardial Infarction; Hypovolemia 1 Nephrolithiasis 2 Pancreatitis 4 Post-operative Vocal Cord Paralysis 1 Hyperemesis Gravidarum; Allergic Rhinitis 1 Pulmonary Edema 2 Pulmonary Edema; Hypertensive Crisis 1 Pulmonary Embolism 5 Pulmonary Embolism; Myocardial Infarction 1 Pulmonary Embolism; Pulmonary Artery Hypertension 1 Pulmonary Fibrosis 2 Pulmonary Mass 1 Reactive Arthritis 1 Rhabdomyolysis 1 Ruptured Aneurysm; Hypovolemic Shock 1 Severe Aortic Stenosis 1 Small Bowel Obstruction 1 Stills Disease 1 Pulmonary Artery Hypertension; Congestive Heart Failure 1 Systemic Lupus Erythematosis 1 Tracheobronchomalacia 1 Transient Ischemic Attack 1 Ulcerative Colitis 1 Urethral Obstruction 1 ^(a)This patient was adjudicated as having a bacterial ARI with Bacillus species identified as the etiologic agent. We later recognized Bacillus species was not the correct microbiological etiology although the clinical history was otherwise consistent with bacterial pneumonia. As this error was identified after model derivation, we included the subject in all subsequent analyses.

TABLE 9 Probes selected for the Bacterial ARI, Viral ARI, and Non-infectious Illness Classifiers. Probe names are presented as Affymetrix probe IDs. Values for each probe represent the weight of each probe in the specified classifier. Affymetrix Bacterial Viral Non-Infectious Illness Probe ID ARI Classifier ARI Classifier Classifier Gene Symbol RefSeq ID Gene Name 200042_at 0 0.038998 0 HSPC117 NM_014306 chromosome 22 open reading frame 28 200947_s_at 1.78944 0 0 GLUD1 NM_005271 glutamate dehydrogenase 1 201055_s_at 0 0 1.25363 HNRPA0 NM_006805 heterogeneous nuclear ribonucleoprotein A0 201188_s_at 0.606326 0 0 ITPR3 NM_002224 inositol 1,4,5-triphosphate receptor, type 3 201341_at 0.109677 0 0 ENC1 NM_003633 ectodermal-neural cortex (with BTB-like domain) 202005_at −0.68053 0 0 ST14 NM_021978 suppression of tumorigenicity 14 (colon carcinoma) 202145_at 0 0.166043 0 LY6E NM_002346; NM_001127213 lymphocyte antigen 6 complex, locus E 202284_s_at −0.35646 0 0 CDKN1A NM_078467; NM_000389 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 202411_at −0.05224 0 0 IFI27 NM_005532; NM_001130080 interferon, alpha-inducible protein 27 202509_s_at 0 0 0.416714 TNFAIP2 NM_006291 tumor necrosis factor, alpha-induced protein 2 202644_s_at 0.340624 0 0 TNFAIP3 NM_006290 tumor necrosis factor, alpha-induced protein 3 202688_at 0 0.005084 0 TNFSF10 NM_003810 tumor necrosis factor (ligand) superfamily, member 10 202709_at 0.427849 0 0 FMOD NM_002023 fibromodulin 202720_at 0 0.07874 0 TES NM_152829; NM_015641 testis derived transcript (3 LIM domains) 202864_s_at 0 0.02937 0 SP100 NM_003113; NM_001080391 SP100 nuclear antigen 202973_x_at −0.11208 0 0 FAM13A1 NM_014883; NM_001015045 family with sequence similarity 13, member A 203045_at −0.8509 0 0 NINJ1 NM_004148 ninjurin 1 203153_at −0.13374 0 0 IFIT1 NM_001548 interferon-induced protein with tetratricopeptide repeats 1 203275_at 0 0.074576 0 IRF2 NM_002199 interferon regulatory factor 2 203313_s_at −1.09463 0 0 TGIF NM_173211; NM_173210; TGFB-induced factor homeobox 1 NM_003244; NM_174886; NM_173209; NM_173208; NM_173207; NM_170695 203392_s_at 0 −0.01392 0 CTBP1 NM_001328; NM_001012614 C-terminal binding protein 1 203455_s_at 0 0 −0.0805395 SAT NM_002970 spermidine/spermine N1-acetyltransferase 1 203882_at 0 0.034534 0 ISGF3G NM_006084 interferon regulatory factor 9 203979_at −0.00999 0 0.301178 CYP27A1 NM_000784 cytochrome P450, family 27, subfamily A, polypeptide 1 204392_at 0 0.111394 0 CAMK1 NM_003656 calcium/calmodulin-dependent protein kinase I 204490_s_at 0.007328 0 0 CD44 NM_000610; NM_001001389; CD44 molecule (Indian blood group) NM_001001390; NM_001001391; NM_001001392 204545_at 0.342478 0 0 PEX6 NM_000287 peroxisomal biogenesis factor 6 204750_s_at 0.537475 0 0 DSC2 NM_004949; NM_024422 desmocollin 2 205001_s_at 0 −0.06712 0 DDX3Y NM_001122665; NM_004660 DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked 205008_s_at 0 0.223868 0 CIB2 NM_006383 calcium and integrin binding family member 2 205033_s_at 0 −0.08786 0 DEFA1 /// DEFA3 NM_004084; NM_001042500 defensin, alpha 1 205048_s_at −0.01145 0 0 PSPH NM_004577 phosphoserine phosphatase-like; phosphoserine phosphatase 205098_at −0.11641 0 0 CCR1 NM_001295 chemokine (C-C motif) receptor 1 205153_s_at 0.132886 0 0 CD40 NM_152854; NM_001250 CD40 molecule, TNF receptor superfamily member 5 205164_at 0.46638 0 0 GCAT NM_014291; NM_001171690 glycine C-acetyltransferase (2-amino-3-ketobutyrate coenzyme A ligase) 205200_at 0.87833 0 0 CLEC3B NM_003278 C-type lectin domain family 3, member B 205312_at 0 0 −0.394304 SPI1 NM_001080547; NM_003120 spleen focus forming virus (SFFV) proviral integration oncogene spi1 206207_at −0.08529 0 0 CLC NM_001828 Charcot-Leyden crystal protein 206371_at 0.043902 0 0 FOLR3 NM_000804 folate receptor 3 (gamma) 206647_at 0.065039 0 0 HBZ NM_005332 hemoglobin, zeta 206676_at 0 0 0.0774651 CEACAM8 NM_001816 carcinoembryonic antigen-related cell adhesion molecule 8 206896_s_at 0.482822 0 0 GNG7 NM_052847 guanine nucleotide binding protein (G protein), gamma 7 206918_s_at 1.00926 0 0 CPNE1 NM_152929; NM_152928; RNA binding motif protein 12; copine I NM_152927; NM_003915; NM_152931; NM_152930; NM_006047; NM_152925; NM_152926; NM_152838 206934_at 0.151959 0 0 SIRPB1 NM_001135844; NM_006065; signal-regulatory protein beta 1 NM_001083910 207075_at −0.06273 0 0 CIAS1 NM_004895; NM_001079821; NLR family, pyrin domain containing 3 NM_001127462; NM_001127461; NM_183395 207194_s_at 0.3162 0 0 ICAM4 NM_022377; NM_001544; intercellular adhesion molecule 4 (Landsteiner-Wiener blood NM_001039132 group) 207244_x_at 1.30636 0 0 CYP2A6 NM_000762 cytochrome P450, family 2, subfamily A, polypeptide 6 207606_s_at 0.299775 0 0 ARHGAP12 NM_018287 Rho GTPase activating protein 12 207718_x_at 0.039296 0 0 CYP2A6 /// CYP2A7 /// NM_000764; NM_030589 cytochrome P450, family 2, subfamily A, polypeptide 7 CYP2A7P1 /// CYP2A13 207840_at 0 0.118889 0 CD160 NM_007053 CD160 molecule 207860_at 0.376517 0 0 NCR1 NM_001145457; NM_001145458; natural cytotoxicity triggering receptor 1 NM_004829 208029_s_at −0.02051 0 0.394049 LAPTM4B NM_018407 lysosomal protein transmembrane 4 beta 208545_x_at 0.265408 0 0 TAF4 NM_003185 TAF4 RNA polymerase II, TATA box binding protein (TBP)- associated factor, 135 kDa 208601_s_at −0.27058 0 0 TUBB1 NM_030773 tubulin, beta 1 208702_x_at 0 0 0.0426262 APLP2 NM_001642; NM_001142277; amyloid beta (A4) precursor-like protein 2 NM_001142278; NM_001142276 208736_at 0 0.582264 −0.0862941 ARPC3 NM_005719 similar to actin related protein 2/3 complex subunit 3; hypothetical LOC729841; actin related protein 2/3 complex, subunit 3, 21 kDa 208886_at 0.149103 0 0 H1F0 NM_005318 H1 histone family, member 0 208974_x_at 0 0.742946 0 KPNB1 NM_002265 karyopherin (importin) beta 1 209031_at 0 0 0.237916 IGSF4 NM_014333; NM_001098517 cell adhesion molecule 1 209360_s_at 0.303561 0 0 RUNX1 NM_001122607; NM_001001890; runt-related transcription factor 1 NM_001754 209396_s_at 0 0 0.0355749 CHI3L1 NM_001276 chitinase 3-like 1 (cartilage glycoprotein-39) 209511_at 0 −0.03119 0 POLR2F NM_021974 polymerase (RNA) II (DNA directed) polypeptide F 209605_at −0.49934 0 0 TST NM_003312 thiosulfate sulfurtransferase (rhodanese) 209919_x_at 0.613197 0 0 GGT1 XM_001129425; NM_013430; gamma-glutamyltransferase light chain 3; gamma- NM_001032365; NM_005265; glutamyltransferase 4 pseudogene; gamma- NM_001032364; XM_001129377 glutamyltransferase 2; gamma-glutamyltransferase 1; gamma- glutamyltransferase light chain 5 pseudogene 210365_at 0.576935 0 0 RUNX1 NM_001122607; NM_001001890; runt-related transcription factor 1 NM_001754 210724_at 0 0 0.482166 EMR3 NM_032571 egf-like module containing, mucin-like, hormone receptor-like 3 210797_s_at 0 0.185097 0 OASL NM_198213; NM_003733 2′-5′-oligoadenylate synthetase-like 212035_s_at 2.0241 0 −1.26034 EXOC7 NM_001145298; NM_001145299; exocyst complex component 7 NM_015219; NM_001145297; NM_001145296; NM_001013839 212162_at 0 −0.01023 0 KIDINS220 NM_020738 kinase D-interacting substrate, 220 kDa 212657_s_at 0 0 −0.254507 IL1RN NM_000577; NM_173841; interleukin 1 receptor antagonist NM_173842; NM_173843 212697_at 0 0 −1.02451 LOC162427 NM_178126 family with sequence similarity 134, member C 212708_at 0.032564 0 0 LOC339287 NM_001012241 male-specific lethal 1 homolog (Drosophila) 212914_at 0 0 0.0099678 CBX7 NM_175709 chromobox homolog 7 212947_at 0.286979 0 0 SLC9A8 NM_015266 solute carrier family 9 (sodium/hydrogen exchanger), member 8 213223_at 0.686657 0 0 RPL28 NM_001136134; NM_000991; ribosomal protein L28 NM_001136137; NM_001136135; NM_001136136 213300_at −0.5783 0 0 KIAA0404 NM_015104 ATG2 autophagy related 2 homolog A (S. cerevisiae) 213573_at 0 0 −0.497655 KPNB1 NM_002265 karyopherin (importin) beta 1 213633_at −1.01336 0 0 SH3BP1 NM_018957 SH3-domain binding protein 1 214085_x_at −0.36761 0 0 GLIPR1 NM_006851 GLI pathogenesis-related 1 214097_at 0.00915 −0.5768 0 RPS21 NM_001024 ribosomal protein S21 214175_x_at 0 0 −0.266628 PDLIM4 NM_003687; NM_001131027 PDZ and LIM domain 4 214326_x_at −0.69811 0 0.261075 JUND NM_005354 jun D proto-oncogene 214582_at 0 0 0.0377349 PDE3B NM_000922 phosphodiesterase 3B, cGMP-inhibited 214617_at −0.26196 0 0 PRF1 NM_005041; NM_001083116 perforin 1 (pore forming protein) 214800_x_at 0 0.103261 0 BTF3 /// LOC345829 NM_001037637; NM_001207 basic transcription factor 3; basic transcription factor 3, like 1 pseudogene 214955_at −0.10065 0 0 TMPRSS6 NM_153609 transmembrane protease, serine 6 215184_at 0 −0.06503 0 DAPK2 NM_014326 death-associated protein kinase 2 215268_at 0.038178 0 0 KIAA0754 NM_015038 hypothetical LOC643314 215606_s_at 0.479765 0 0 RAB6IP2 NM_178040; NM_015064; ELKS/RAB6-interacting/CAST family member 1 NM_178037; NM_178038; NM_178039 215804_at 1.94364 0 0 EPHA1 NM_005232 EPH receptor A1 215848_at 0 0.326241 0 ZNF291 NM_001145923; NM_020843 S-phase cyclin A-associated protein in the ER 216289_at 0 −0.00075 0 XM_002347085; XM_002342934; G protein-coupled receptor 144 XM_002346195; NM_001161808 216303_s_at 0.31126 0 0 MTMR1 NM_003828 myotubularin related protein 1 216473_x_at 0 −0.0343 0 DUX4 /// LOC399839 /// XM_927996; XM_001720078; double homeobox, 4-like; similar to double homeobox 4c; LOC401650 /// XM_001722088; NM_001164467; similar to double homeobox, 4; double homeobox, 4 LOC440013 /// XM_928023; XM_495858; LOC440014 /// XM_941455; NM_001127386; LOC440015 /// XM_001720082; XM_001720798; LOC440016 /// XM_496731; NM_001127387; LOC440017 /// XM_495854; XM_495855; LOC441056 NM_001127388; NM_033178; NM_001127389; XM_001724713 216713_at 0.510039 0 0 KRIT1 NM_194454; NM_001013406; KRIT1, ankyrin repeat containing NM_004912; NM_194456; NM_194455 216867_s_at −0.05347 0 0 PDGFA NM_033023; NM_002607 platelet-derived growth factor alpha polypeptide 217143_s_at −0.3891 0 0 TRA@ /// TRD@ ambiguous (pending) 217408_at 0 1.07798 −0.0690681 MRPS18B NM_014046 mitochondrial ribosomal protein S18B 217593_at −0.07475 0 0 ZNF447 NM_001145542; NM_001145543; zinc finger and SCAN domain containing 18 NM_001145544; NM_023926 217717_s_at 0.638943 0 0 YWHAB NM_139323; NM_003404 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide 218095_s_at 0 −0.61377 0 TPARL NM_018475 transmembrane protein 165 218306_s_at 0 0 0.784894 HERC1 NM_003922 hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 218595_s_at 0 0 −0.411708 HEATR1 NM_018072 HEAT repeat containing 1 218812_s_at −0.96799 0 0 C7orf19 NM_032831; NM_001126340 ORAI calcium release-activated calcium modulator 2 219055_at −0.08524 0 0 FLJ10379 NM_018079 S1 RNA binding domain 1 219066_at 0 0.221446 0 PPCDC NM_021823 phosphopantothenoylcysteine decarboxylase 219130_at 0 −0.15077 0 FLJ10287 NM_019083 coiled-coil domain containing 76 219382_at 0.866643 0 0 SERTAD3 NM_013368; NM_203344 SERTA domain containing 3 219437_s_at 0 −0.40545 0.198273 ANKRD11 XM_001720760; NM_013275; ankyrin repeat domain 11; hypothetical protein XM_001721661; XM_001721649 LOC100128265 219523_s_at 0 0 −0.0236667 ODZ3 NM_001080477 odz, odd Oz/ten-m homolog 3 (Drosophila) 219777_at 0 0.25509 0 GIMAP6 NM_024711 GTPase, IMAP family member 6 220059_at −0.86817 0 0 BRDG1 NM_012108 signal transducing adaptor family member 1 220122_at 0.399475 0 0 MCTP1 NM_024717; NM_001002796 multiple C2 domains, transmembrane 1 220308_at 0 −0.03456 0 CCDC19 NM_012337 coiled-coil domain containing 19 221491_x_at −0.65143 0 0 HLA-DRB1 /// HLA-DRB3 XM_002346768; NM_022555; major histocompatibility complex, class II, DR beta 3 /// HLA-DRB4 XM_002346769 221874_at −0.40581 0 0.017015 KIAA1324 NM_020775 KIAA1324 222059_at 0 −0.11226 0 ZNF335 NM_022095 zinc finger protein 335 44673_at −0.0308 0 0 SN NM_023068 sialic acid binding Ig-like lectin 1, sialoadhesin 216571_at 0.878426 0 0 NM_000543; NM_001007593 sphingomyelin phosphodiesterase 1, acid lysosomal 216943_at −0.91643 0 0 207436_x_at 0 0.243737 0 KIAA0894 ambiguous (pending)

TABLE 10 Genes in the Bacterial ARI, Viral ARI, and Non-infectious Illness (NI) Classifiers, grouped by biologic process. Gene accession numbers are provided in Table 9. Biologic process Bacterial Viral NI Cell cycle regulation JUND* (−), NINJ1, IFI27, ZNF291 JUND* (+) CDKN1A, C7orf19, SERTAD3 Regulation of cell growth YWHAB, PDGFA APLP2 Development/ GLIPR1, RUNX1, ST14, TGIF, CTBP1 SP1, CEACAM8, ODZ3 Differentiation EPHA1 RNA transcription, FLJ10379, RPS21* (+), DDX3Y, POLR2F, RPS21* HEATR1, MRPS18B* (−) processing RPL28, TAF4, RPP25 (−), BTF3, MRPS18B* (+), HSPC117, FLJ10287 Role in nuclear transport KPNB1 KPNB1 Role in cell and membrane RAB6IP2, SH3BP1, EXOC7* TPARL EXOC7* (−), HERC1, trafficking (+), LAPTM4B, CPNE1, LAPTM4B, KIAA1324, GNG7, TPARL, KIAA1324 APLP2 Cell structure/adhesion TMPRSS6, TUBB1, TES, ARPC3* (+), PDLIM4, IGSF4, PDE3B, ARHGAP12, ICAM4, DSC2, KIDINS220 ARPC3* (−), CHI3L1 FMOD Role in cell stress response KIAA1324, KRIT1, ENC1 CBX7, APLP2, KIAA1324 Role in autophagy LAPTM4B* (−), KIAA1324* (−) KIAA1324* (+), LAPTM4B* (+) Role in apoptosis KRIT1, GLIPR1, CIAS1 DAPK2, TNFSF10 General Inflammatory TNFA1P3, FMOD, ITPR3, TNFSF10 HNRPAO, EMR3, IL1RN, response CIAS1, GNG7, CLC, IFI27, TNFAIP2, CHI3L1 CCR1 Interferon response IFIT1 SP100, IRF2, OASL, ISGF3G Cytotoxic response PRF1 DefA1/3 Toxin response P450 gene cluster, CYP2A6, ENC1, GGT1, TST T-cell signaling TRA/D@, CD44 Ly6E, CAMK1, CD160 B-cell signaling BRDG1, HLA-DRB1/3/4, CD40 NK-cell response NCR1 CD160 Phospholipid and calcium MTMR1, CPNE1, PSPH, signaling ITPR3, CLC, MCTP1 Fatty acid metabolism PEX6, GLUD1 Cholesterol metabolism CYP27A1* (−) CYP27A1* (+) Amino acid metabolism GLUD1, PSPH, GCAT *Genes listed in more than one classifier. In cases where such overlapping genes have different directions of expression, increased expression is denoted by (+) and decreased expression is denoted by (−).

Example 3 The Bacterial/Viral/SIRS Assay Contemplated on a TLDA Platform

We will develop a custom multianalyte, quantitative real-time PCR (RT-PCR) assay on the 384-well TaqMan Low Density Array (TLDA, Applied Biosystems) platform. TLDA cards will be manufactured with one or more TaqMan primer/probe sets specific for a gene mRNA transcript in the classifier(s) in each well, along with multiple endogenous control RNA targets (primer/probe sets) for data normalization. For each patient sample, purified total RNA is reverse transcribed into cDNA, loaded into a master well and distributed into each assay well via centrifugation through microfluidic channels. TaqMan hydrolysis probes rely on 5′ to 3′ exonuclease activity to cleave the dual-labeled probe during hybridization to complementary target sequence with each amplification round, resulting in fluorescent signal production. In this manner, quantitative detection of the accumulated PCR products in “real-time” is possible. During exponential amplification and detection, the number of PCR cycles at which the fluorescent signal exceeds a detection threshold is the threshold cycle (C_(t)) or quantification cycle (C_(q))—as determined by commercial software for the RT-PCR instrument. To quantify gene expression, the C_(t) for a target RNA is subtracted from the C_(t) of endogenous normalization RNA (or the geometric mean of multiple normalization RNAs), providing a deltaC_(t) value for each RNA target within a sample which indicates relative expression of a target RNA normalized for variability in amount or quality of input sample RNA or cDNA.

The data for the quantified gene signatures are then processed using a computer and according to the probit classifier described above (equation 1) and reproduce here. Normalized gene expression levels of each gene of the signature are the explanatory or independent variables or features used in the classifier, in this example the general form of the classifier is a probit regression formulation:

P(having condition)=ψ(β₁ X ₁+β₂ X ₂+ . . . +β_(d) X _(d))   (equation 1)

where the condition is bacterial ARI, viral ARI, or non-infection illness; Φ(⋅) is the probit link function; {β₁,β₂, . . . , β_(d)} are the coefficients obtained during training; {X₁,X₂, . . . , X_(d)} are the normalized genes expression values of the signature; and d is the size of the signature (number of genes). The value of the coefficients for each explanatory variable are specific to the technology platform used to measure the expression of the genes or a subset of genes used in the probit regression model. The computer program computes a score, or probability, and compares the score to a threshold value. The sensitivity, specificity, and overall accuracy of each classifier is optimized by changing the threshold for classification using receiving operating characteristic (ROC) curves.

A preliminary list of genes for the TLDA platform based on the signature from the Affymetrix platform (Affy signature) as well as from other sources is provided below in Table 1A. Weights appropriate for the TLDA platform for the respective classifiers were thereafter determined as described below in Example 4.

TABLE 1A Preliminary list of genes for development of classifiers for TLDA platform. Alternate Non- TLDA assay Original Affy ID Affy ID GROUP Bacterial Viral infectious GENE identifier 219437_s_at 212332_at Affy signature — — — ANKRD11 Hs00331872_s1 208702_x_at 201642_at Affy signature — — — APLP2 Hs00155778_m1 207606_s_at 212633_at Affy signature — — — ARHGAP12 Hs00367895_m1 201659_s_at 209444_at Affy signature — — — ARL1 Hs01029870_m1 208736_at 201132_at Affy signature — — — ARPC3 Hs00855185_g1 205965_at 218695_at Affy signature — — — BATF Hs00232390_m1 214800_x_at 209876_at Affy signature — — — BTF3 Hs00852566_g1 209031_at 209340_at Affy signature — — — CADM1 Hs00296064_s1 204392_at 214054_at Affy signature — — — CAMK1 Hs00269334_m1 201949_x_at 37012_at Affy signature — — — CAPZB Hs00191827_m1 207840_at 213830_at Affy signature — — — CD160 Hs00199894_m1 200663_at 203234_at Affy signature — — — CD63 Hs00156390_m1 220935_s_at 219271_at Affy signature — — — CDK5RAP2 Hs01001427_m1 206676_at 207269_at Affy signature — — — CEACAM8 Hs00266198_m1 209396_s_at 209395_at Affy signature — — — CHI3L1 Hs01072230_g1 205008_s_at 58900_at Affy signature — — — CIB2 Hs00197280_m1 205200_at 206034_at Affy signature — — — CLEC3B Hs00162844_m1 203979_at 49111_at Affy signature — — — CYP27A1 Hs01017992_g1 207244_x_at 209280_at Affy signature — — — CYP2A13 Hs00711162_s1 215184_at 217521_at Affy signature — — — DAPK2 Hs00204888_m1 205001_s_at 214131_at Affy signature — — — DDX3Y Hs00965254_gH 205033_s_at 207269_at Affy signature — — — DEFA3 Hs00414018_m1 204750_s_at 205418_at Affy signature — — — DSC2 Hs00951428_m1 216473_x_at 221660_at Affy signature — — — DUX4 Hs03037970_g1 210724_at 220246_at Affy signature — — — EMR3 Hs01128745_m1 215804_at 206903_at Affy signature — — — EPHA1 Hs00975876_g1 212035_s_at 200935_at Affy signature — — — EXOC7 Hs01117053_m1 212697_at 46665_at Affy signature — — — FAM134C Hs00738661_m1 209919_x_at 218695_at Affy signature — — — GGT1 Hs00980756_m1 219777_at 202963_at Affy signature — — — GIMAP6 Hs00226776_m1 200947_s_at 202126_at Affy signature — — — GLUD1 Hs03989560_s1 218595_s_at 217103_at Affy signature — — — HEATR1 Hs00985319_m1 218306_s_at 212232_at Affy signature — — — HERC1 Hs01032528_m1 221491_x_at 203290_at Affy signature — — — HLA-DRB3 Hs00734212_m1 201055_s_at 37012_at Affy signature — — — HNRNPA0 Hs00246543_s1 203153_at 219863_at Affy signature — — — IFIT1 Hs01911452_s1 214022_s_at 35254_at Affy signature — — — IFITM1 Hs00705137_s1 212657_s_at 202837_at Affy signature — — — IL1RN Hs00893626_m1 203275_at 213038_at Affy signature — — — IRF2 Hs01082884_m1 203882_at 201649_at Affy signature — — — IRF9 Hs00196051_m1 215268_at 200837_at Affy signature — — — KIAA0754 Hs03055204_s1 221874_at 203063_at Affy signature — — — KIAA1324 Hs00381767_m1 213573_at 31845_at Affy signature — — — KPNB1 Hs00158514_m1 208029_s_at 212573_at Affy signature — — — LAPTM4B Hs00363282_m1 202145_at 204972_at Affy signature — — — LY6E Hs03045111_g1 220122_at 218323_at Affy signature — — — MCTP1 Hs01115711_m1 217408_at 212846_at Affy signature — — — MRPS18B Hs00204096_m1 207860_at 212318_at Affy signature — — — NCR1 Hs00950814_g1 203045_at 213038_at Affy signature — — — NINJ1 Hs00982607_m1 210797_s_at 205660_at Affy signature — — — OASL Hs00984390_m1 214175_x_at 204600_at Affy signature — — — PDGFA Hs00184792_m1 219066_at 217497_at Affy signature — — — PPCDC Hs00222418_m1 214617_at 212070_at Affy signature — — — PRF1 Hs00169473_m1 218700_s_at 203816_at Affy signature — — — RAB7L1 Hs00187510_m1 215342_s_at 218695_at Affy signature — — — RABGAP1L Hs02567906_s1 219143_s_at 204683_at Affy signature — — — RPP25 Hs00706565_s1 214097_at 201094_at Affy signature — — — RPS21 Hs00963477_g1 210365_at 222307_at Affy signature — — — SAT1 Hs00971739_g1 215848_at 81811_at Affy signature — — — SCAPER Hs02569575_s1 212900_at 204496_at Affy signature — — — SEC24A Hs00378456_m1 44673_at 219211_at Affy signature — — — SIGLEC1 Hs00988063_m1 201802_at 206361_at Affy signature — — — SLC29A1 Hs01085704_g1 202864_s_at 202863_at Affy signature — — — SP100 Hs00162109_m1 205312_at 205707_at Affy signature — — — SPI1 Hs00231368_m1 202005_at 205418_at Affy signature — — — ST14 Hs04330394_g1 220059_at 202478_at Affy signature — — — STAP1 Hs01038134_m1 219523_s_at 206903_at Affy signature — — — TENM3 Hs01111787_m1 202720_at 201344_at Affy signature — — — TES Hs00210319_m1 203313_s_at 212232_at Affy signature — — — TGIF1 Hs00820148_g1 218095_s_at 219157_at Affy signature — — — TMEM165 Hs00218461_m1 202509_s_at 212603_at Affy signature — — — TNFAIP2 Hs00196800_m1 219130_at 200685_at Affy signature — — — TRMT13 Hs00219487_m1 208601_s_at 205127_at Affy signature — — — TUBB1 Hs00258236_m1 217717_s_at 205037_at Affy signature — — — YWHAB Hs00793604_m1 217593_at 222141_at Affy signature — — — ZSCAN18 Hs00225073_m1 213300_at 219014_at Affy signature — — — ATG2A Hs00390076_m1 212914_at 211938_at Affy signature — — — CBX7 Hs00545603_m1 220308_at 202452_at Affy signature — — — CCDC19 Hs01099244_m1 205098_at 213361_at Affy signature — — — CCR1 Hs00928897_s1 205153_s_at 215346_at Affy signature — — — CD40 Hs01002913_g1 204490_s_at 205026_at Affy signature — — — CD44 Hs00153304_m1 202284_s_at 213324_at Affy signature — — — CDKN1A Hs00355782_m1 206207_at 206361_at Affy signature — — — CLC Hs01055743_m1 206918_s_at 200964_at Affy signature — — — CPNE1 Hs00537765_m1 203392_s_at 222265_at Affy signature — — — CTBP1 Hs00972289_g1 207718_x_at 44702_at Affy signature — — — CYP2A6 Hs00711162_s1 207718_x_at 44702_at Affy signature — — — CYP2A7 Hs00711162_s1 201341_at 209717_at Affy signature — — — ENC1 Hs00171580_m1 215606_s_at 211999_at Affy signature — — — ERC1 Hs00327390_s1 202973_x_at 201417_at Affy signature — — — FAM13A Hs01040170_m1 202709_at 222265_at Affy signature — — — FMOD Hs00157619_m1 206371_at 205844_at Affy signature — — — FOLR3 Hs01549264_m1 205164_at 209391_at Affy signature — — — GCAT Hs00606568_gH 214085_x_at 203799_at Affy signature — — — GLIPR1 Hs00199268_m1 206896_s_at 206126_at Affy signature — — — GNG7 Hs00192999_m1 216289_at 206338_at Affy signature — — — GPR144 Hs01369282_m1 208886_at 213096_at Affy signature — — — H1F0 Hs00961932_s1 206647_at 40850_at Affy signature — — — HBZ Hs00744391_s1 207194_s_at 218225_at Affy signature — — — ICAM4 Hs00169941_m1 202411_at 213797_at Affy signature — — — IFI27 Hs01086373_g1 201188_s_at 213958_at Affy signature — — — ITPR3 Hs00609948_m1 212162_at 210148_at Affy signature — — — KIDINS220 Hs01057000_m1 216713_at 213049_at Affy signature — — — KRIT1 Hs01090981_m1 212708_at 202897_at Affy signature — — — MSL1 Hs00290567_s1 216303_s_at 222265_at Affy signature — — — MTMR1 Hs01021250_m1 207075_at 203906_at Affy signature — — — NLRP3 Hs00366465_m1 214582_at 222317_at Affy signature — — — ORAI2 Hs01057217_m1 216867_s_at 202909_at Affy signature — — — PDE38 Hs00236997_m1 204545_at 320_at Affy signature — — — PDLIM4 Hs00165457_m1 209511_at 218333_at Affy signature — — — POLR1C Hs00191646_m1 209511_at 218333_at Affy signature — — — POLR2F Hs00222679_m1 213633_at 204632_at Affy signature — — — PSG4 Hs00978711_m1 213633_at 204632_at Affy signature — — — PSG4 Hs01652476_m1 205048_s_at 203303_at Affy signature — — — PSPH Hs00190154_m1 213223_at 210607_at Affy signature — — — RPL28 Hs00357189_g1 200042_at 212247_at Affy signature — — — RTCB Hs00204783_m1 209360_s_at 203916_at Affy signature — — — RUNX1 Hs00231079_m1 219382_at 209575_at Affy signature — — — SERTAD3 Hs00705989_s1 213633_at 204632_at Affy signature — — — SH3BP1 Hs00978711_m1 213633_at 204632_at Affy signature — — — SH3BP1 Hs01652476_m1 206934_at 202545_at Affy signature — — — SIRPB1 Hs01092173_m1 212947_at 220404_at Affy signature — — — SLC9A8 Hs00905708_m1 216571_at 202396_at Affy signature — — — SMPD1 Hs01086851_m1 219055_at 219439_at Affy signature — — — SRBD1 Hs01005222_m1 208545_x_at 204600_at Affy signature — — — TAF4 Hs01122669_m1 214955_at 217162_at Affy signature — — — TMPRSS6 Hs00541789_s1 202644_s_at 55692_at Affy signature — — — TNFAIP3 Hs01568119_m1 202688_at 219684_at Affy signature — — — TNFSF10 Hs00234356_m1 209605_at 212897_at Affy signature — — — TST Hs04187383_m1 222059_at 216076_at Affy signature — — — ZNF335 Hs00223060_m1 202509_s_at NA InTxAlternate — — — TNFAIP2 Hs00969305_m1 202672_s_at NA PanViralArray — — — ATF3 Hs00910173_m1 218943_s_at NA PanViralArray — — — DDX58 Hs01061436_m1 219863_at NA PanViralArray — — — HERC5 Hs01061821_m1 214059_at NA PanViralArray — — — IFI44 Hs00951349_m1 204439_at NA PanViralArray — — — IFI44L Hs00915294_g1 204415_at NA PanViralArray — — — IFI6 Hs00242571_m1 203153_at NA PanViralArray — — — IFIT1 Hs03027069_s1 217502_at NA PanViralArray — — — IFIT2 Hs01922738_s1 204747_at NA PanViralArray — — — IFIT3 Hs01922752_s1 205483_s_at NA PanViralArray — — — ISG15 Hs01921425_s1 205569_at NA PanViralArray — — — LAMP3 Hs00180880_m1 202145_at NA PanViralArray — — — LY6E Hs03045111_g1 202086_at NA PanViralArray — — — MX1 Hs00182073_m1 205552_s_at NA PanViralArray — — — OAS1 Hs00973637_m1 202869_at NA PanViralArray — — — OAS2 Hs00973637_m1 218400_at NA PanViralArray — — — OAS3 Hs00934282_g1 205660_at NA PanViralArray — — — OASL Hs00984390_m1 213797_at NA PanViralArray — — — RSAD2 Hs00369813_m1 219684_at NA PanViralArray — — — RTP4 Hs00223342_m1 210657_s_at NA PanViralArray — — — SEPT4 Hs00910209_g1 200986_at NA PanViralArray — — — SERPING1 Hs00934330_m1 222154_s_at NA PanViralArray — — — SPATS2L Hs01016364_m1 206026_s_at NA PanViralArray — — — TNFAIP6 Hs01113602_m1 219211_at NA PanViralArray — — — USP18 Hs00276441_m1 206133_at NA PanViralArray — — — XAF1 Hs01550142_m1 NA NA Reference — — — FPGS Hs00191956_m1 NA NA Reference — — — PPIB Hs00168719_m1 NA NA Reference — — — TRAP1 Hs00972326_m1 NA NA Reference — — — DECR1 Hs00154728_m1 NA NA Reference — — — GAPDH Hs99999905_m1 NA NA Reference — — — 18S Hs99999901_s1 NA 203799_at Replacement — — — CD302 Hs00208436_m1 NA 31845_at Replacement — — — ELF4 Hs01086126_m1 NA 204600_at Replacement — — — EPHB3 Hs01082563_g1 NA 206903_at Replacement — — — EXOG Hs01035290_m1 NA 218695_at Replacement — — — EXOSC4 Hs00363401_g1 NA 212232_at Replacement — — — FNBP4 Hs01553131_m1 NA 209876_at Replacement — — — GIT2 Hs00331902_s1 NA 204683_at Replacement — — — ICAM2 Hs01015796_m1 NA 201642_at Replacement — — — IFNGR2 Hs00985251_m1 NA 203799_at Replacement — — — LY75-CD302 Hs00208436_m1 NA 209280_at Replacement — — — MRC2 Hs00195862_m1 NA 212603_at Replacement — — — MRPS31 Hs00960912_m1 NA 221660_at Replacement — — — MYL10 Hs00540809_m1 NA 203290_at Replacement — — — PEX6 Hs00165457_m1 NA 201417_at Replacement — — — SOX4 Hs00268388_s1 NA 44702_at Replacement — — — SYDE1 Hs00973080_m1 NA 222261_at Replacement — — — TLDC1 Hs00297285_m1 NA 202452_at Replacement — — — ZER1 Hs01115240_m1

Example 4 Bacterial/Viral/SIRS Classification Using Gene Expression Measured by RT-qPCR Implemented on the TLDA Platform

The genes of the three signatures that compose the Host Response-ARI (HR-ARI) test were transitioned to a Custom TaqMan® Low Density Array Cards from ThermoFisher Scientific (Waltham, Mass.). Expression of these gene signatures were measured using custom multianalyte quantitative real time PCR (RT-qPCR) assays on the 384-well TaqMan Low Density Array (TLDA; Thermo-Fisher) platform. TLDA cards were designed and manufactured with one or more TaqMan primer/probe sets per well, each representing a specific RNA transcript in the ARI signatures, along with multiple endogenous control RNA targets (TRAP1, PPIB, GAPDH, FPGS, DECR1 and 18S) that are used to normalize for RNA loading and to control for plate-to-plate variability. In practice, two reference genes (out of five available), which have the smallest coefficient of variation across samples for the normalization procedure, were selected and primer/probe sets with more than 33% missing values (below limits of quantification) were discarded. The remaining missing values (if any), are set to 1+max(Cq), where Cq is the quantification cycle for RT-qPCR. Normalized expression values were then calculated as the average of the selected references minus the observed Cq values for any given primer/probe set. See Hellemans et al. (2007) Genome Biol 2007; 8(2):R19.

A total of 174 unique primer/probe sets were assayed per sample. Of these primer/probes, 144 primer/probe sets measure gene targets representative of the 132 previously described Affymetrix (microarray) probes of the three ARI gene signatures (i.e., the genes in the bacterial gene expression signature, the viral gene expression signature and the non-infectious gene expression signature); 6 probe sets are for reference genes, and we additionally assayed 24 probe sets from a previously-discovered pan-viral gene signature. See U.S. Pat. No. 8,821,876; Zaas et al. Cell Host Microbe (2009) 6(3):207-217. In addition, a number of primer/probe sets for “replacement” genes were added for training, the expression of these genes being correlated with the expression of some genes from the Affymetrix signature. Some genes are replaced because the RT-qPCR assays for these genes, when performed using TLDA probes, did not perform well.

For each sample, total RNA was purified from PAXgene Blood RNA tubes (PreAnalytix) and reverse transcribed into cDNA using the Superscript VILO cDNA synthesis kit (Thermo-Fisher) according to the manufacturer's recommended protocol. A standard amount of cDNA for each sample was loaded per master well, and distributed into each TaqMan assay well via centrifugation through microfluidic channels. The TaqMan hydrolysis probes rely on 5′ to 3′ exonuclease activity to cleave the dual-labeled probe during hybridization to complementary target sequence with each amplification round, resulting in fluorescent signal production. Quantitative detection of the fluorescence indicates accumulated PCR products in “real-time.” During exponential amplification and detection, the number of PCR cycles at which the fluorescent signal exceeds a detection threshold is the threshold cycle (C_(t)) or quantification cycle (C_(q))—as determined by commercial software for the RT-qPCR instrument.

Sample/Cohort Selection:

Under an IRB-approved protocol, we enrolled patients presenting to the emergency department with acute respiratory illness (See Table 11, below). The patients in this cohort are a subset of those reported in Table 1 of Tsalik et al. (2016) Sci Transl Med 9(322):1-9, which is incorporated by reference herein. Retrospective clinical adjudication of the clinical and other test data for these patients leads to one of three assignments: bacterial ARI, viral ARI, or non-infectious illness.

TABLE 11 Demographic information for the enrolled cohort Number Mean age, # Samples (Viral/ of Gender years Ethnicity Bacterial/Non- Cohort subject^(a) (M/F) (Range)^(b) (B/W/O) Admitted Infectious Illness) Enrolled 317 122/151 45 (6-88)  135/116/22 61% 115/70/88 Derivation Cohort Viral 115 44/71 45 (6-88)  40/59/16 21% Bacterial 70 35/35 49 (14-88) 46/22/2 94% Non-infectious 88 43/45 49 (14-88) 49/35/4 88% Illness^(c) Healthy 44 23/21 30 (20-59) 8/27/6^(d) 0% ^(a)Only subjects with viral, bacterial, or non-infectious illness were included (when available) from each validation cohort. ^(b)When mean age was unavailable or could not be calculated, data is presented as either Adult or Pediatric. ^(c)Non-infectious illness was defined by the presence of SIRS criteria, which includes at least two of the following four features; Temperature <36° or >38° C.; Heart rate >90 beats per minute; Respiratory rate >20 breaths per minute or arterial partial pressure of CO₂ <32 mmHg; and white blood cell count <4000 or >12,000 cells/mm³ or >10% band form neutrophils. ^(d)Three subjects did not report ethnicity. M, Male. F, Female. B, Black. W, White, O, Other/Unknown. GSE numbers refer to NCBI Gene Expression Omnibus datasets. N/A, Not available based on published data.

Data Analysis Methods:

During the data preprocessing stage, we select a subset of at least two reference gene targets (out of five available) with the smallest coefficient of variation across samples and plates. We discard targets with more than 33% missing values (17 targets below the limit of quantification), only if these values are not over represented in any particular class, e.g., bacterial ARI. Next we impute the remaining missing values to 1+max(C_(q)), then normalize the expression values for all targets using the reference combination previously selected. In particular, we compute normalized expression values as the mean of the selected references (DECR1 and PPIB) minus the C_(q) values of any given target.

Once the data has been normalized, we proceed to build the classification model by fitting a sparse logistic regression model to the data (Friedman et al. (2010) J. Stat. Softw. 33, 1-22). This model estimates the probability that a subject belongs to a particular class as a weighted sum of normalized gene targets. Specifically, we write, p(subject is of class)=σ(w₁x₁+ . . . +w_(p)x_(p)), where σ is the logistic function, w₁, . . . , w_(p) are classification weights estimated during the fitting procedure, x₁, . . . , x_(p) represent the p gene targets containing normalized expression values.

Similar to the array-based classifier, we build three binary classifiers: (1) bacterial ARI vs. viral ARI and non-infectious illness; (2) viral ARI vs. bacterial ARI and non-infectious illness; and (3) non-infectious illness vs. bacterial and viral ARI. After having fitted the three classifiers, we have estimates for p(bacterial ARI), p(viral ARI) and p(non-infectious illness). The thresholds for each of the classifiers are selected from Receiving Operating Characteristic (ROC) curves using a symmetric cost function (expected sensitivity and specificity are approximately equal) (Fawcett (2006) Pattern Recogn Lett 27:861-874). As a result, a subject is predicted as bacterial ARI if p(bacterial ARI)>t_(b), where t_(b) is the threshold for the bacterial ARI classifier. We similarly select thresholds for the viral ARI and non-infectious illness classifiers, t_(v) and t_(n), respectively. If desired, a combined prediction can be made by selecting the most likely condition, i.e., the one with largest probability, specifically we write, argmax{p(bacterial ARI),p(viral ARI),p(non-infectious illness)}.

Results:

During the initial transition of the microarray-discovered genomic classifiers onto the TLDA platform, we assayed 32 samples that also had been assayed by microarray. This group served to confirm that TLDA-based RT-qPCR measurement of the gene transcripts that compose the ARI classifier recapitulates the results obtained for microarray-based measurement of gene transcripts, and is therefore a valid methodology for classifying patients as having bacterial or viral ARI, or having non-infectious illness. We found that from the 32 samples tested both on TLDA and microarray platforms, when assessed using their corresponding classifiers, there is agreement of 84.4%, which means that 27 of 32 subjects had the same combined prediction in both microarray and TLDA-based classification models.

After demonstrating concordance between microarray and TLDA-based classification, we tested an additional 63 samples, using the TLDA-based classification, from patients with clinical adjudication of ARI status but without previously-characterized gene expression patterns. In total, therefore, 95 samples were assessed using the TLDA-based classification test. This dataset from 95 samples allowed us to evaluate how the TLDA-based RT-qPCR platform classifies new patients, using only the clinical adjudication as the reference standard. In this experiment, we observed an overall accuracy of 81.1%, which corresponds to 77/95 correctly classified samples. More specifically, the model yielded bacterial ARI, viral ARI, and non-infectious illness accuracies of 80% (24 correct of 30), 77.4% (24 correct of 31) and 85.3% (29 correct of 34), respectively. In terms of the performance of the individual classifiers, we observed area under the ROC curves of 0.92, 0.86 and 0.91, for the bacterial ARI, viral ARI and non-infectious illness classifier, respectively. Provided that we do not count with a validation dataset for any of the classifiers, yet we want unbiased estimates of classification performance (accuracies and areas under the ROC curve), we are reporting leave-one-out cross-validated performance metrics.

The weights and thresholds for each of the classifiers (bacterial ARI, viral ARI and non-infectious illness) are shown in the Table 12, shown below. Note that this Table lists 151 gene targets instead of 174 gene targets because the reference genes were removed in the preprocessing stage, as described above, as were 17 targets for which there were missing values. These 17 targets were also removed during the preprocessing stage.

If the panviral signature genes are removed, we see a slight decreased performance, no larger than 5% across AUC, accuracies and percent of agreement values.

Summary:

The composite host-response ARI classifier is composed of gene expression signatures that are diagnostic of bacterial ARI versus viral ARI, versus non-infectious illness and a mathematical classification framework. The mathematical classifiers provide three discrete probabilities: that a subject has a bacterial ARI, viral ARI, or non-infectious illness. In each case, a cutoff or threshold may be specified above which threshold one would determine that a patient has the condition. In addition, one may modify the threshold to alter the sensitive and specificity of the test.

The measurement of these gene expression levels can occur on a variety of technical platforms. Here, we describe the measurement of these signatures using a TLDA-based RT-qPCR platform. Moreover, the mathematical framework that determines ARI etiology probabilities is adapted to the platform by platform-specific training to accommodate transcript measurement methods (i.e., establishing platform-specific weights, w₁, . . . , w_(p)). Similar, straightforward, methodology could be conducted to translate the gene signatures to other gene expression detection platforms, and then train the associated classifiers. This Example also demonstrates good concordance between TLDA-based and microarray-based classification of etiology of ARI. Finally, we show the use of the TLDA-based RT-qPCR platform and associated mathematical classifier to diagnose new patients with acute respiratory illness.

TABLE 12 Genes, TLDA probe/primers, and classifier weights for the bacterial, viral and non-infectious illness classifiers. TLDA Assay ID Bacterial Viral Non-infectious Group Gene Symbol RefSeq ID Gene Name Hs00153304_m1 0.44206 −0.19499 0 CD44 NM_000610.3; NM_001202555.1; hCG1811182 Celera Annotation; CD44 molecule (Indian blood NM_001001392.1; NM_001202556.1; group) NM_001001391.1; NM_001001390.1; NM_001001389.1 Hs00155778_m1 0 0 0 APLP2 NM_001142278.1; NM_001142277.1; hCG2032871 Celera Annotation; amyloid beta (A4) precursor-like NM_001142276.1; NR_024515.1; protein 2 NR_024516.1; NM_001642.2; NM_001243299.1 Hs00156390_m1 0.07707 −0.15022 0 CD63 NM_001780.5; NM_001267698.1; CD63 molecule; hCG20743 Celera Annotation NM_001257389.1; NM_001257390.1; NM_001257391.1 Hs00158514_m1 0 0 0 KPNB1 NM_002265.5 hCG1773668 Celera Annotation; karyopherin (importin) beta 1 Hs00162109_m1 0 0.012558 0 SP100 NM_003113.3; NM_001080391.1; SP100 nuclear antigen; hCG34336 Celera Annotation NM_001206702.1; NM_001206703.1; NM_001206701.1; NM_001206704.1 Hs00165457_m1 0.14396 −0.00784 0 PEX6 NM_000287.3 peroxisomal biogenesis factor 6; hCG17647 Celera Annotation Hs00169473_m1 0 −0.04883 0.135154 PRF1 NM_005041.4; NM_001083116.1 hCG22817 Celera Annotation; perforin 1 (pore forming protein) Hs00169941_m1 0 −0.33225 0 ICAM4 NM_001544.4; NM_022377.3 intercellular adhesion molecule 4 (Landsteiner-Wiener blood group); hCG28480 Celera Annotation Hs00171580_m1 0 −0.04133 0 ENC1 NM_001256575.1; NM_001256576.1; hCG37104 Celera Annotation; ectodermal-neural cortex 1 (with NM_003633.3; NM_001256574.1 BTB domain) Hs00187510_m1 0.38204 −0.19399 −0.242396 RAB7L1 NM_001135662.1; NM_003929.2 hCG19156 Celera Annotation; RAB7; member RAS oncogene family-like 1 Hs00190154_m1 0.0726 0 −0.128456 PSPH NM_004577.3 phosphoserine phosphatase; hCG1811513 Celera Annotation Hs00191827_m1 0 0 0 CAPZB NM_001282162.1; NM_004930.4 capping protein (actin filament) muscle Z-line; beta; hCG41078 Celera Annotation Hs00192999_m1 0.08266 0 −0.127277 GNG7 NM_052847.2 guanine nucleotide binding protein (G protein); gamma 7; hCG20107 Celera Annotation Hs00196051_m1 0.05 −0.4723 0 IRF9 NM_006084.4 interferon regulatory factor 9; hCG40171 Celera Annotation Hs00196800_m1 0 0 0 TNFAIP2 NM_006291.2 tumor necrosis factor; alpha-induced protein 2; hCG22889 Celera Annotation Hs00197280_m1 −0.14204 0.089619 0.147283 CIB2 NM_006383.3; NM_001271888.1 calcium and integrin binding family member 2; hCG38933 Celera Annotation Hs00199268_m1 0 −0.10536 0.38895 GLIPR1 NM_006851.2 hCG26513 Celera Annotation; GLI pathogenesis-related 1 Hs00199894_m1 0 −0.10571 0.02064 CD160 NR_103845.1; NM_007053.3 hCG1762288 Celera Annotation; CD160 molecule Hs00204096_m1 0 0 0 MRPS18B NM_014046.3 hCG2039591 Celera Annotation; mitochondrial ribosomal protein S18B Hs00204783_m1 −0.12369 0.330219 0 RTCB NM_014306.4 RNA 2′; 3′-cyclic phosphate and 5′-OH ligase; hCG41412 Celera Annotation Hs00204888_m1 0 0 0 DAPK2 NM_014326.3 death-associated protein kinase 2; hCG32392 Celera Annotation Hs00210319_m1 0 0.061489 0 TES NM_015641.3; NM_152829.2 testis derived transcript (3 LIM domains); hCG39086 Celera Annotation Hs00218461_m1 0.18667 0 −0.125865 TMEM165 NR_073070.1; NM_018475.4 hCG20603 Celera Annotation; transmembrane protein 165 Hs00219487_m1 0.32643 0 −0.350154 TRMT13 NM_019083.2 hCG31836 Celera Annotation; tRNA methyltransferase 13 homolog (S. cerevisiae) Hs00222418_m1 −0.08795 0.254466 0 PPCDC NM_021823.3 phosphopantothenoylcysteine decarboxylase; hCG21917 Celera Annotation Hs00222679_m1 0 0.072372 0 POLR2F; NM_021974.3 polymerase (RNA) II (DNA directed) polypeptide F; hCG41858 LOC100131530 Celera Annotation; uncharacterized LOC100131530 Hs00223060_m1 0 −0.12877 0.034889 ZNF335 NM_022095.3 zinc finger protein 335; hCG40026 Celera Annotation Hs00225073_m1 0 0.661155 −0.183337 ZSCAN18 NM_001145544.1; NM_001145543.1; hCG201365 Celera Annotation; zinc finger and SCAN domain NM_023926.4; NM_001145542.1 containing 18 Hs00226776_m1 0 0.198622 −0.254653 GIMAP6 NM_001244072.1; NM_001244071.1; hCG1655100 Celera Annotation; GTPase; IMAP family member 6 NM_024711.5 Hs00231079_m1 0.0787 0 −0.089259 RUNX1 NM_001001890.2; NM_001754.4 runt-related transcription factor 1; hCG2007747 Celera Annotation Hs00231368_m1 0.30434 0 −0.130472 SPI1 NM_001080547.1; NM_003120.2 spleen focus forming virus (SFFV) proviral integration oncogene; hCG25181 Celera Annotation Hs00232390_m1 0.22771 −0.39445 0 BATF NM_006399.3 hCG22346 Celera Annotation; basic leucine zipper transcription factor; ATF-like Hs00234356_m1 0 0 −0.005804 TNFSF10 NR_033994.1; NM_003810.3 tumor necrosis factor (ligand) superfamily; member 10; hCG20249 Celera Annotation Hs00246543_s1 0 0.096747 0 HNRNPA0 NM_006805.3 hCG1639951 Celera Annotation; heterogeneous nuclear ribonucleoprotein A0 Hs00258236_m1 0 0.067758 −0.014686 TUBB1 NM_030773.3 tubulin; beta 1 class VI; hCG28550 Celera Annotation Hs00259863_m1 −0.03861 0.156335 0 ORAI2 NM_001126340.2; NM_001271818.1; hCG1736771 Celera Annotation; ORAI calcium release-activated NM_032831.3 calcium modulator 2 Hs00266198_m1 −0.03709 0.174789 0 CEACAM8 NM_001816.3 carcinoembryonic antigen-related cell adhesion molecule 8; hCG21882 Celera Annotation Hs00269334_m1 0 0.11804 −0.054795 CAMK1 NM_003656.4 calcium/calmodulin-dependent protein kinase I; hCG21548 Celera Annotation Hs00290567_s1 0.10454 −0.57285 0 MSL1 NM_001012241.1 hCG31740 Celera Annotation; male-specific lethal 1 homolog (Drosophila) Hs00296064_s1 −0.11096 0.162636 0 CADM1 NM_014333.3; NM_001098517.1 cell adhesion molecule 1 Hs00327390_s1 −0.27728 0.219012 0.023246 ERC1 NM_178040.2; NR_027949.1; ELKS/RAB6-interacting/CAST family member 1 NR_027946.1; NR_027948.1; NM_178039.2 Hs00331872_s1 0 −0.04877 0 ANKRD11 NM_013275.5; NM_001256182.1; hCG1980824 Celera Annotation; ankyrin repeat domain 11 NM_001256183.1 Hs00355782_m1 0 0 0 CDKN1A NM_001220778.1; NM_001220777.1; cyclin-dependent kinase inhibitor 1A (p21; Cip1); hCG15367 Celera NM_000389.4; NM_078467.2 Annotation Hs00357189_g1 0 0 0 RPL28 NM_001136137.1; NM_000991.4; ribosomal protein L28; hCG38234 Celera Annotation NM_001136134.1; NM_001136135.1; NM_001136136.1 Hs00363282_m1 0 −0.39826 0.298323 LAPTM4B NM_018407.4 lysosomal protein transmembrane 4 beta; hCG2008559 Celera Annotation Hs00366465_m1 0 0 0 NLRP3 NM_001127461.2; NM_001079821.2; NLR family; pyrin domain containing 3; hCG1982559 Celera NM_001243133.1; NM_004895.4; Annotation NM_001127462.2; NM_183395.2 Hs00367895_m1 0 0 0 ARHGAP12 NM_001270698.1; NM_001270697.1; Rho GTPase activating protein 12; hCG2017264 Celera Annotation NM_018287.6; NM_001270699.1; NM_001270696.1; NM_001270695.1 Hs00378456_m1 0 0 0 SEC24A NM_021982.2; NM_001252231.1 SEC24 family; member A (S. cerevisiae); hCG1981418 Celera Annotation Hs00381767_m1 −0.08167 −0.02155 0.251085 KIAA1324 NR_049774.1; NM_020775.4; hCG1997600 Celera Annotation; KIAA1324 NM_001267049.1; NM_001267048.1 Hs00390076_m1 −0.4019 0 0.306895 ATG2A NM_015104.2 hCG2039982 Celera Annotation; autophagy related 2A Hs00414018_m1 0 0 0 DEFA3; DEFA1; NM_004084.3; NM_005217.3; defensin; alpha 3; neutrophil-specific; defensin; alpha 1; defensin; DEFA1B NM_001042500.1 alpha 1B Hs00537765_m1 0.12016 0 −0.311567 CPNE1 NM_001198863.1; NM_152926.2; copine I; hCG38213 Celera Annotation NR_037188.1; NM_152927.2; NM_152925.2; NM 152928.2; NM_003915.5 Hs00541789_s1 0 0 0 TMPRS56 NM_153609.2 hCG2011224 Celera Annotation; transmembrane protease; serine 6 Hs00545603_m1 −0.15652 0 0.157219 CBX7 NM_175709.3 chromobox homolog 7; hCG41710 Celera Annotation Hs00606568_gH 0 0.024977 0 GCAT NM_014291.3; NM_001171690.1 hCG41842 Celera Annotation; glycine C-acetyltransferase Hs00609948_m1 −0.1261 0 0.132035 ITPR3 NM_002224.3 hCG40301 Celera Annotation; inositol 1; 4; 5-trisphosphate receptor; type 3 Hs00705137_s1 0 0.190805 −0.207955 IFITM1 NM_003641.3 interferon induced transmembrane protein 1; hCG1741134 Celera Annotation Hs00705989_s1 0 0.264586 −0.237834 SERTAD3 NM_203344.2; NM_013368.3 SERTA domain containing 3; hCG201413 Celera Annotation Hs00706565_s1 0 0.247956 −0.127891 RPP25 NM_017793.2 ribonuclease P/MRP 25 kDa subunit; hCG1643228 Celera Annotation Hs00711162_s1 −0.01602 0.105815 0 CYP2A13; NM_000764.2; NM_030589.2; cytochrome P450; family 2; subfamily A; polypeptide 13; CYP2A7; NM 000766.4; NM_000762.5 cytochrome P450; family 2; subfamily A; polypeptide 7; CYP2A6 cytochrome P450; family 2; subfamily A; polypeptide 6; hCG2039740 Celera Annotation; hCG1780445 Celera Annotation Hs00734212_m1 0.03633 −0.10881 0 HLA-DRB3; NM_022555.3 hCG2001518 Celera Annotation; major histocompatibility complex; HLA-DRB1 class II; DR beta 3; major histocompatibility complex; class II; DR beta 1 Hs00738661_m1 −0.2813 0 0.255274 FAM134C NR_026697.1; NM_178126.3 family with sequence similarity 134; member C; hCG2043027 Celera Annotation Hs00793604_m1 0 0 −0.392469 YWHAB NM_003404.4; NM_139323.3 hCG38378 Celera Annotation; tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein; beta polypeptide Hs00820148_g1 0 0 0.082524 TGIF1 NM_173207.2; NM_003244.3; TGFB-induced factor homeobox 1; hCG1994498 Celera Annotation NM_001278682.1; NM_170695.3; NM_001278686.1; NM_001278684.1; NM_173210.2; NM_173209.2; NM_173208.2; NM_174886.2; NM_173211.1 Hs00852566_g1 0 0 0.090784 BTF3 NM_001207.4; NM_001037637.1 hCG37844 Celera Annotation; basic transcription factor 3 Hs00855185_g1 0.22884 −0.16129 0 ARPC3 NM_001278556.1; NM_005719.2 hCG1787850 Celera Annotation; hCG1730237 Celera Annotation; actin related protein 2/3 complex; subunit 3; 21 kDa Hs00893626_m1 0 0 −0.131321 IL1RN NM_000577.4; NM_173841.2; hCG1733963 Celera Annotation; interleukin 1 receptor antagonist NM_173842.2; NM_173843.2 Hs00905708_m1 0 0 0 SLC9A8 NM_001260491.1; NR_048537.1; solute carrier family 9; subfamily A (NHE8; cation proton antiporter NR_048538.1; NR_048539.1; 8); member 8; hCG37890 Celera Annotation NR_048540.1; NM_015266.2 Hs00928897_s1 0 0 0 CCR1 NM_001295.2 hCG15324 Celera Annotation; chemokine (C-C motif) receptor 1 Hs00950814_g1 0 0 0.035502 NCR1 NM_001145457.2; NM_001242356.2; hCG19670 Celera Annotation; natural cytotoxicity triggering NM_004829.6 receptor 1 Hs00951428_m1 0 0.113402 0 DSC2 NM_024422.3; NM_004949.3 hCG24896 Celera Annotation; desmocollin 2 Hs00961932_s1 0 0 0 H1F0 NM_005318.3 hCG1641126 Celera Annotation; H1 histone family; member 0 Hs00963477_g1 0 −0.00884 0 RPS21 NM_001024.3 hCG41768 Celera Annotation; ribosomal protein S21 Hs00971739_g1 0 0.128754 0 SAT1 NR_027783.1; NM_002970.2 hCG17885 Celera Annotation; spermidine/spermine N1- acetyltransferase 1 Hs00972289_g1 −0.36317 0.301793 0.148178 CTBP1 NM_001012614.1; NM_001328.2 hCG1981976 Celera Annotation; C-terminal binding protein 1 Hs00978711_m1 0 −0.19534 0.079881 SH3BP1 NM_018957.3 hCG41861 Celera Annotation; SH3-domain binding protein 1 Hs00980756_m1 0 −0.27613 0.042497 GGT1 NM_001032364.2; NM_001032365.2; gamma-glutamyltransferase 1; hCG2010666 Celera Annotation NM_005265.2; NM_013430.2 Hs00982607_m1 0 0 0 NINJ1 NM_004148.3 ninjurin 1; hCG18015 Celera Annotation Hs00984390_m1 0 0.074028 −0.022201 OASL NM_198213.2; NM_003733.3 hCG27362 Celera Annotation; 2′-5′-oligoadenylate synthetase-like Hs00985319_m1 −0.01147 0.079048 0 HEATR1 NM_018072.5 HEAT repeat containing 1; hCG25461 Celera Annotation Hs00988063_m1 −0.08452 0.168519 0 SIGLEC1 NM_023068.3 hCG39260 Celera Annotation; sialic acid binding Ig-like lectin 1; sialoadhesin Hs01001427_m1 0.04332 −0.60556 0 CDK5RAP2 NR_073558.1; NR_073554.1; hCG27455 Celera Annotation; CDK5 regulatory subunit associated NR_073555.1; NR_073556.1; protein 2 NM_001272039.1; NR_073557.1; NM_001011649.2; NM_018249.5 Hs01002913_g1 0 0 0 CD40 NM_152854.2; NM_001250.4 hCG40016 Celera Annotation; CD40 molecule; TNF receptor superfamily member 5 Hs01005222_m1 0 0.326033 0 SRBD1 NM_018079.4 S1 RNA binding domain 1; hCG1987258 Celera Annotation Hs01017992_g1 0 0 0.179899 CYP27A1 NM_000784.3 hCG15569 Celera Annotation; cytochrome P450; family 27; subfamily A; polypeptide 1 Hs01021250_m1 0.01799 0.196899 −0.140181 MTMR1 NM_003828.2 hCG1640369 Celera Annotation; myotubularin related protein 1 Hs01029870_m1 0 −0.58215 0.22929 ARL1 NM_001177.4 hCG1782029 Celera Annotation; ADP-ribosylation factor-like 1 Hs01032528_m1 0 −0.36595 0.410577 HERC1 NM_003922.3 hCG1818283 Celera Annotation; HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 Hs01038134_m1 −0.13717 0.004773 0.049685 STAP1 NM_012108.2 signal transducing adaptor family member 1; hC640344 Celera Annotation Hs01040170_m1 0.04344 −0.17845 −0.052769 FAM13A NM_014883.3; NM_001265578.1; hCG39059 Celera Annotation; family with sequence similarity 13; NM_001015045.2; NM_001265580.1; member A NM_001265579.1 Hs01055743_m1 −0.30697 0 0.257693 CLC NM_001828.5 hCG43348 Celera Annotation; Charcot-Leyden crystal galectin Hs01057000_m1 0 −0.68353 0.082116 KIDINS220 NM_020738.2 hCG23067 Celera Annotation; kinase D-interacting substrate; 220 kDa Hs01057217_m1 −0.45125 0.327746 0.070281 PDE3B NM_000922.3 phosphodiesterase 3B; cGMP-inhibited; hCG23682 Celera Annotation Hs01072230_g1 0 −0.00364 0.169878 CHI3L1 NM_001276.2 chitinase 3-like 1 (cartilage glycoprotein-39); hCG24326 Celera Annotation Hs01082884_m1 0.29147 −0.1223 0 IRF2 NM_002199.3 hCG16244 Celera Annotation; interferon regulatory factor 2 Hs01085704_g1 0 0 0 SLC29A1 NM_001078174.1; NM_004955.2; hCG19000 Celera Annotation; solute carrier family 29 (equilibrative NM_001078177.1; NM_001078176.2; nucleoside transporter); member 1 NM_001078175.2 Hs01086373_g1 −0.11199 0.274551 −0.063877 IFI27 NM_005532.3; NM_001130080.1 interferon; alpha-inducible protein 27; hCG22330 Celera Annotation Hs01086851_m1 0.37999 −0.28298 0 SMPD1 NM_001007593.2; NM_000543.4 sphingomyelin phosphodiesterase 1; acid lysosomal; hCG24080 Celera Annotation Hs01090981_m1 0 0 0 KRIT1 NM_194456.1; NM_194454.1; hCG1812017 Celera Annotation; KRIT1; ankyrin repeat containing NM_004912.3; NM_001013406.1; NM_194455.1 Hs01092173_m1 0.09825 0 0 SIRPB1 NM_001083910.2; NM_006065.3 signal-regulatory protein beta 1; hCG39419 Celera Annotation Hs01099244_m1 0.01588 −0.22063 0.055484 CCDC19 NM_012337.2 hCG39740 Celera Annotation; coiled-coil domain containing 19 Hs01115711_m1 0.2568 0 −0.127859 MCTP1 NM_001002796.2; NM_024717.4 multiple C2 domains; transmembrane 1; hCG1811111 Celera Annotation Hs01117053_m1 0 0 0 EXOC7 NR_028133.1 exocyst complex component 7; hCG40887 Celera Annotation Hs01122669_m1 0 −0.03893 0.066177 TAF4 NM_003185.3 hCG41771 Celera Annotation; TAF4 RNA polymerase II; TATA box binding protein (TBP)-associated factor; 135 kDa Hs01128745_m1 0 0.031228 0 EMR3 NM_032571.3 hCG95683 Celera Annotation; egf-like module containing; mucin- like; hormone receptor-like 3 Hs01549264_m1 0.02825 −0.12496 0 NM_000804.2 hCG1640300 Celera Annotation; folate receptor 3 (gamma) Hs01568119_m1 0 0.181259 −0.076525 TNFAIP3 NM_001270508.1; NM_006290.3; hCG16787 Celera Annotation; tumor necrosis factor; alpha-induced NM_001270507.1 protein 3 Hs01911452_s1 0 0 0 IFIT1 NM 001548.4; NM_001270928.1; hCG24571 Celera Annotation; interferon-induced protein with NM_001270927.1; NM_001270930.1; tetratricopeptide repeats 1 NM_001270929.1 Hs02567906_s1 −0.22881 0.019641 0 RABGAP1L NM_001243763.1; NM_014857.4; hCG2024869 Celera Annotation; RAB GTPase activating protein 1- NM_001035230.2 like Hs02569575_s1 0 0 −0.12916 SCAPER NM_001145923.1; NM_020843.2 hCG40799 Celera Annotation; S-phase cyclin A-associated protein in the ER Hs03037970_g1 0 0 0 DUX4L7; NM_001278056.1; NM_001164467.2; double homeobox 4 like 7; double homeobox 4 like 5; double DUX4L5; NR_038191.1; NM 001177376.2; homeobox 2; double homeobox 4 like 2; double homeobox 4 like DUX4L6; NM_012147.4; NM_001127389.2; 6; double homeobox 4; double homeobox protein 4-like; double DUX4L2; DUX2; NM_001127388.2; NM_001127387.2; homeobox 4-like; double homeobox 4 like 4; double homeobox 4 DUX4; NM_033178.4; NM_001127386.2 like 3 LOC100653046; DUX4L; DUX4L4; DUX4L3 Hs03045111_g1 −0.02913 0.054676 0 LY6E NM_002346.2; NM_001127213.1 hCG1765592 Celera Annotation; lymphocyte antigen 6 complex; locus E Hs03055204_s1 0 0 0 KIAA0754 NM_015038.1 KIAA0754 Hs03989560_s1 −0.28689 0.169135 0.040358 GLUD1 NM_005271.3 glutamate dehydrogenase 1 Hs04187383_m1 0 0 0 TST NM 003312.5; NM_001270483.1 thiosulfate sulfurtransferase (rhodanese); hCG41451 Celera Annotation Hs00969305_m1 0 −0.50526 0 InTxAlternate TNFAIP2 NM_006291.2 tumor necrosis factor; alpha-induced protein 2; hCG22889 Celera Annotation Hs00180880_m1 0 0 0 PanViral LAMP3 NM_014398.3 lysosomal-associated membrane protein 3; hCG16067 Celera Annotation Hs00182073_m1 0 0.043305 0 PanViral MX1 NM_002462.3; NM_001144925.1; myxovirus (influenza virus) resistance 1; interferon-inducible NM_001178046.1 protein p78 (mouse); hCG401239 Celera Annotation Hs00213443_m1 0 0.009468 −0.051318 PanViral OAS2 NM_016817.2 2′-5′-oligoadenylate synthetase 2; 69/71 kDa; hCG38536 Celera Annotation Hs00223342_m1 0 0 0 PanViral RTP4 NM_022147.2 hCG1653633 Celera Annotation; receptor (chemosensory) transporter protein 4 Hs00242571_m1 0 0 −0.078103 PanViral IFI6 NM_022873.2; NM_002038.3; interferon; alpha-inducible protein 6; hCG1727099 Celera NM_022872.2 Annotation Hs00276441_m1 0 0.033981 −0.048548 PanViral USP18 NM_017414.3 ubiquitin specific peptidase 18; hCG21533 Celera Annotation Hs00369813_m1 −0.02854 0 0 PanViral RSAD2 NM_080657.4 hCG23898 Celera Annotation; radical S-adenosyl methionine domain containing 2 Hs00910173_m1 0 0.065635 −0.003951 PanViral ATF3 NM_001030287.3; NM_001206484.2; hCG37734 Celera Annotation; activating transcription factor 3 NM_001206488.2; NM_001674.3 Hs00910209_g1 −0.00172 0.07212 0 PanViral SEP4 NM_080416.2; NM_004574.3; septin 4; hCG30696 Celera Annotation NM_001256822.1; NM_080415.2; NM_001256782.1; NR_037155.1; NM_001198713.1 Hs00915294_g1 0 0 0 PanViral IFI44L NM_006820.2 hCG24062 Celera Annotation; interferon-induced protein 44-like Hs00934282_g1 0 0 0 PanViral OAS3 NM_006187.2 2′-5′-oligoadenylate synthetase 3; 100 kDa; hCG40370 Celera Annotation Hs00934330_m1 0 0.065027 0 PanViral SERPING1 NM_000062.2; NM_001032295.1 serpin peptidase inhibitor; clade G (C1 inhibitor); member 1; hCG39766 Celera Annotation Hs00951349_m1 0 0 0 PanViral IFI44 NM_006417.4 interferon-induced protein 44; hCG24065 Celera Annotation Hs00973637_m1 0 0 −0.060351 PanViral OAS1 NM_001032409.1; NM_016816.2; 2′-5′-oligoadenylate synthetase 1; 40/46 kDa; hCG40366 Celera NM_002534.2 Annotation Hs01016364_m1 0 0 0 PanViral SPATS2L NM_001100422.1; NM_015535.2; spermatogenesis associated; serine-rich 2-like; hCG1811464 Celera NM_001100424.1; NM_001100423.1 Annotation Hs01061436_m1 0 0.01828 −0.042268 PanViral DDX58 NM_014314.3 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58; hCG1811781 Celera Annotation Hs01061821_m1 0 0 0 PanViral HERC5 NM_016323.3 HECT and RLD domain containing E3 ubiquitin protein ligase 5; hCG1813153 Celera Annotation Hs01113602_m1 0.05847 0 −0.206842 PanViral TNFAIP6 NM_007115.3 hCG41965 Celera Annotation; tumor necrosis factor; alpha-induced protein 6 Hs01550142_m1 0 −0.06086 0 PanViral XAF1 NR_046398.1; NM_199139.2; hCG1777063 Celera Annotation; XIAP associated factor 1 NM_017523.3; NR_046396.1; NR_046397.1 Hs01921425_s1 0 0.018167 −0.032153 PanViral ISG15 NM_005101.3 ISG15 ubiquitin-like modifier; hCG1771418 Celera Annotation Hs01922738_s1 −0.0409 0.185197 −0.007029 PanViral IFIT2 NM_001547.4 interferon-induced protein with tetratricopeptide repeats 2; hCG1643352 Celera Annotation Hs01922752_s1 0 0 0 PanViral IFIT3 NM_001549.4; NM_001031683.2 hCG24570 Celera Annotation; interferon-induced protein with tetratricopeptide repeats 3 Hs03027069_s1 −0.00733 0 0 PanViral IFIT1 NM_001548.4; NM_001270928.1; interferon-induced protein with tetratricopeptide repeats 1; NM_001270927.1; NM_001270930.1; hCG24571 Celera Annotation NM_001270929.1 Hs00191646_m1 0 0 0 Replacement POLR1C NM_203290.2 polymerase (RNA) I polypeptide C; 30 kDa; hCG18995 Celera Annotation Hs00208436_m1 0 0.013116 0 Replacement CD302; LY75- NM_014880.4; NM_001198763.1; CD302 molecule; hCG40834 Celera Annotation; LY75-CD302 CD302 NM_001198760.1; NM_001198759.1 readthrough Hs00297285_m1 0 −0.46905 0 Replacement TLDC1 NM_020947.3 TBC/LysM-associated domain containing 1; hCG39793 Celera Annotation Hs00331902_s1 0 −0.45598 0.236611 Replacement GIT2 NM_057170.3; NM_014776.3; hCG38510 Celera Annotation; G protein-coupled receptor kinase NM_001135213.1; NM_001135214.1; interacting ArfGAP 2 NM_057169.3 Hs00363401_g1 0 0 −0.077823 Replacement EXOSC4 NM_019037.2 hCG1747868 Celera Annotation; exosome component 4 Hs00960912_m1 0 0.26766 0 Replacement MRPS31 NM_005830.3 mitochondrial ribosomal protein S31; hCG32763 Celera Annotation Hs00985251_m1 0.10711 −0.17404 0 Replacement IFNGR2 NM_005534.3 interferon gamma receptor 2 (interferon gamma transducer 1); hCG401179 Celera Annotation Hs01015796_m1 0 0.189857 0 Replacement ICAM2 NM_001099786.1; NM_001099787.1; intercellular adhesion molecule 2; hCG41817 Celera Annotation NM_001099788.1; NM_001099789.1; NM_000873.3 Hs01035290_m1 −0.05606 0.248968 0 Replacement EXOG NM_005107.3; NM_001145464.1 endo/exonuclease (5′-3′); endonuclease G-like; hCG40337 Celera Annotation Hs01086126_m1 0 0 0 Replacement ELF4 NM_001421.3; NM_001127197.1 E74-like factor 4 (ets domain transcription factor); hCG21000 Celera Annotation Hs01115240_m1 0 −0.79464 0.589673 Replacement ZER1 NM_006336.3 zyg-11 related; cell cycle regulator; hCG1788209 Celera Annotation Hs01553131_m1 0 −0.26139 0.697495 Replacement FNBP4 NM_015308.2 formin binding protein 4; hCG25190 Celera Annotation

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including definitions, will control.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosures described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims. 

1. A method for making acute respiratory illness classifiers for a platform, wherein the classifiers comprise a bacterial ARI classifier, a viral ARI classifier and a non-infectious illness classifier for the platform, said method comprising: (a) obtaining biological samples from a plurality of subjects known to be suffering from a bacterial acute respiratory infection; (b) obtaining biological samples from a plurality of subjects known to be suffering from a viral acute respiratory infection; (c) obtaining biological samples from a plurality of subjects known to be suffering from a non-infectious illness; (d) measuring on said platform the gene expression levels of a plurality of genes (e.g., all expressed genes or transcriptome, or a subset thereof) in each of said biological samples from steps (a), (b) and (c); (e) normalizing the gene expression levels obtained in step (d) to generate normalized gene expression values; and (f) generating a bacterial ARI classifier, a viral ARI classifier and a non-infectious illness classifier for the platform based upon said normalized gene expression values, to thereby make the acute respiratory illness classifiers for the platform.
 2. The method of claim 1, wherein said measuring comprises or is preceded by one or more steps of: purifying cells from said sample, breaking the cells of said sample, and isolating RNA from said sample.
 3. The method of claim 1, wherein said measuring comprises semi-quantitative PCR and/or nucleic acid probe hybridization.
 4. The method of claim 1, wherein said platform comprises an array platform, a thermal cycler platform (e.g., multiplexed and/or real-time PCR platform), a hybridization and multi-signal coded (e.g., fluorescence) detector platform, a nucleic acid mass spectrometry platform, a nucleic acid sequencing platform, or a combination thereof.
 5. The method of claim 1, wherein said generating comprises iteratively: (i) assigning a weight for each normalized gene expression value, entering the weight and expression value for each gene into a classifier (e.g., a linear regression classifier) equation and determining a score for outcome for each of the plurality of subjects, then (ii) determining the accuracy of classification for each outcome across the plurality of subjects, and then (iii) adjusting the weight until accuracy of classification is optimized, to provide said bacterial ARI classifier, viral ARI classifier and non-infectious illness classifier for the platform, wherein genes having a non-zero weight are included in the respective classifier, and optionally uploading components of each classifier (genes, weights and/or etiology threshold value) onto one or more databases.
 6. The method of claim 5, wherein the classifier is a linear regression classifier and said generating comprises converting a score of said classifier to a probability.
 7. The method according to claim 1 further comprising validating said ARI classifier against a known dataset comprising at least two relevant clinical attributes.
 8. A bacterial ARI classifier made according to the method of claim 1, wherein the bacterial ARI classifier comprises expression levels of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes (measurable, e.g., with oligonucleotide probes homologous to said genes) listed as part of a viral ARI classifier in Table 1, Table 2, Table 9, Table 10 and/or Table
 12. 9. A viral ARI classifier made according to the method of claim 1, wherein the viral classifier comprises expression levels of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes (measurable, e.g., with oligonucleotide probes homologous to said genes) listed as part of a viral ARI classifier in Table 1, Table 2, Table 9, Table 10 and/or Table
 12. 10. A non-infectious illness classifier made according to the method of claim 1, said non-infectious classifier comprises expression levels of 5, 10, 20, 30 or 50, to 80, 100, 150 or 200 of the genes (measurable, e.g., with oligonucleotide probes homologous to said genes) listed as part of a non-infectious illness classifier in Table 1, Table 2, Table 9, Table 10 and/or Table
 12. 11. A method for determining an etiology of an acute respiratory illness in a subject suffering therefrom, or at risk thereof, selected from bacterial, viral and/or non-infectious, comprising: (a) obtaining a biological sample from the subject; (b) measuring on a platform gene expression levels of a pre-defined set of genes (i.e., signature) in said biological sample; (c) normalizing the gene expression levels to generate normalized gene expression values; (d) entering the normalized gene expression values into one or more acute respiratory illness classifiers selected from a bacterial acute respiratory infection (ARI) classifier, a viral ARI classifier and a non-infectious illness classifier, said classifier(s) comprising pre-defined weighting values (i.e., coefficients) for each of the genes of the pre-defined set of genes for the platform, optionally wherein said classifier(s) are retrieved from one or more databases; and (e) calculating an etiology probability for one or more of a bacterial ARI, viral ARI and non-infectious illness based upon said normalized gene expression values and said classifier(s), to thereby determine whether the acute respiratory illness in the subject is bacterial in origin, viral in origin, non-infectious in origin, or some combination thereof.
 12. The method of claim 11, further comprising: (f) comparing the probability to pre-defined thresholds, cut-off values, or ranges of values (e.g., a confidence interval) that indicate likelihood of infection.
 13. The method of claim 11, wherein the subject is suffering from acute respiratory illness symptoms.
 14. The method of claim 11, wherein said subject is suspected of having a bacterial infection or a viral infection.
 15. The method of claim 11, wherein, if the sample does not indicate a likelihood of bacterial ARI, further comprises repeating steps (d) and (e) using only the viral classifier and/or non-infectious classifier, to determine whether the acute respiratory illness in the subject is viral in origin, non-infectious in origin, or a combination thereof.
 16. The method of claim 11, wherein, if the sample does not indicate a likelihood of viral ARI, further comprises repeating steps (d) and (e) using only the bacterial classifier and/or non-infectious classifier, to determine whether the acute respiratory illness in the subject is bacterial in origin, non-infectious in origin, or a combination thereof.
 17. The method of claim 11, wherein, if the sample does not indicate a likelihood of non-infectious illness, further comprises repeating steps (d) and (e) using only the bacterial classifier and/or viral classifier, to determine whether the acute respiratory illness in the subject is bacterial in origin, viral in origin, or a combination thereof.
 18. The method of claim 11 in which the method further comprises generating a report assigning the subject a score indicating the probability of the etiology of the acute respiratory illness.
 19. The method as in claim 11 in which the pre-defined set of genes comprises from 30 to 200 genes.
 20. The method according to claim 11 in which the pre-defined set of genes comprises from 30 to 200 genes listed in Table 1, Table 2, Table 9, Table 10 and/or Table
 12. 21. The method as in claim 11 in which the biological sample comprises is selected from the group consisting of peripheral blood, sputum, nasopharyngeal swab, nasopharyngeal wash, bronchoalveolar lavage, endotracheal aspirate, and combinations thereof.
 22. The method as in claim 11 in which the biological sample is a peripheral blood sample.
 23. The method of claim 11, wherein the bacterial acute respiratory infection (ARI) classifier, viral ARI classifier and non-infectious illness classifier are obtained by a method of any one of claims 1-7
 24. A method of treating an acute respiratory illness in a subject comprising administering to said subject an appropriate treatment regimen based on an etiology determined by a method of claim
 11. 25. The method according to claim 24, wherein the appropriate treatment regimen comprises an antibacterial therapy when the etiology is determined to comprise a bacterial ARI.
 26. The method according to claim 24, wherein the appropriate treatment regimen comprises an antiviral therapy when the etiology is determined comprise a viral ARI.
 27. A method of monitoring response to a vaccine or a drug in a subject suffering from or at risk of an acute respiratory illness selected from bacterial, viral and/or non-infectious, comprising determining a host response of said subject, said determining carried out by a method of claim
 11. 28. The method of claim 27, wherein the drug is an antibacterial drug or an antiviral drug.
 29. A system for determining an etiology of an acute respiratory illness in a subject selected from bacterial, viral and/or non-infectious, comprising: at least one processor; a sample input circuit configured to receive a biological sample from the subject; a sample analysis circuit coupled to the at least one processor and configured to determine gene expression levels of the biological sample; an input/output circuit coupled to the at least one processor; a storage circuit coupled to the at least one processor and configured to store data, parameters, and/or classifiers; and a memory coupled to the processor and comprising computer readable program code embodied in the memory that when executed by the at least one processor causes the at least one processor to perform operations comprising: controlling/performing measurement via the sample analysis circuit of gene expression levels of a pre-defined set of genes (i.e., signature) in said biological sample; normalizing the gene expression levels to generate normalized gene expression values; retrieving from the storage circuit a bacterial acute respiratory infection (ARI) classifier, a viral ARI classifier and a non-infectious illness classifier, said classifier(s) comprising pre-defined weighting values (i.e., coefficients) for each of the genes of the pre-defined set of genes; entering the normalized gene expression values into one or more acute respiratory illness classifiers selected from the bacterial acute respiratory infection (ARI) classifier, the viral ARI classifier and the non-infectious illness classifier; calculating an etiology probability for one or more of a bacterial ARI, viral ARI and non-infectious illness based upon said classifier(s); and controlling output via the input/output circuit of a determination whether the acute respiratory illness in the subject is bacterial in origin, viral in origin, non-infectious in origin, or some combination thereof.
 30. The system of claim 29, where said system comprises computer readable code to transform quantitative, or semi-quantitative, detection of gene expression to a cumulative score or probability of the etiology of the ARI.
 31. The system of claim 29, wherein said system comprises an array platform, a thermal cycler platform (e.g., multiplexed and/or real-time PCR platform), a hybridization and multi-signal coded (e.g., fluorescence) detector platform, a nucleic acid mass spectrometry platform, a nucleic acid sequencing platform, or a combination thereof.
 32. The system of claim 29, wherein the pre-defined set of genes comprises from 30 to 200 genes.
 33. The system of claim 29, wherein the pre-defined set of genes comprises from 30 to 200 genes listed in Table 1, Table 2, Table 9, Table 10 and/or Table
 12. 